Mammalian cytokine complexes

ABSTRACT

Purified genes encoding cytokine from a mammal, reagents related thereto including purified proteins, specific antibodies, and nucleic acids encoding this molecule are provided. Methods of using said reagents and diagnostic kits are also provided.

This filing is a conversion to U.S. Utility Patent Application of U.S.Ser. No. 60/124,319, filed Mar. 11, 1999, which is incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention pertains to compositions related to proteins whichfunction in controlling biology and physiology of mammalian cells, e.g.,cells of a mammalian immune system. In particular, it provides purifiedgenes, proteins, antibodies, related reagents, and methods useful, e.g.,to regulate activation, development, differentiation, and function ofvarious cell types, including hematopoietic cells.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to the technique ofintegrating genetic information from a donor source into vectors forsubsequent processing, such as through introduction into a host, wherebythe transferred genetic information is copied and/or expressed in thenew environment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. See, e.g., Paul (1998) Fundamental Immunology (4th ed.) RavenPress, NY. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which will lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system disorders. Some of thesefactors are hematopoietic growth factors, e.g., granulocyte colonystimulating factor (G-CSF). See, e.g., Thomson (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Mire-Sluis and Thorpe (ed.1998) Cytokines Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press, andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, and/ordifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types.

Research to better understand and treat various immune disorders hasbeen hampered by the general inability to maintain cells of the immunesystem in vitro. Immunologists have discovered that culturing thesecells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

From the foregoing, it is evident that the discovery and development ofnew lymphokines, e.g., related to G-CSF and/or IL-6, could contribute tonew therapies for a wide range of degenerative or abnormal conditionswhich directly or indirectly involve the immune system and/orhematopoietic cells. In particular, the discovery and development oflymphokines which enhance or potentiate the beneficial activities ofknown lymphokines would be highly advantageous. The present inventionprovides new interleukin compositions and related compounds, and methodsfor their use.

SUMMARY OF THE INVENTION

The present invention is directed to mammalian, e.g., primate or rodent,interleukin-B60 (IL-B60) and its biological activities. It includesnucleic acids coding for polypeptides themselves and methods for theirproduction and use. The nucleic acids of the invention arecharacterized, in part, by their homology to complementary DNA (cDNA)sequences disclosed herein, and/or by functional assays for growthfactor- or cytokine-like activities, e.g., G-CSF (see Nagata (1994) inThomson The Cytokine Handbook 2d ed., Academic Press, San Diego) and/orIL-6 (see Hirano (1994) in Thomson The Cytokine Handbook 2d ed.,Academic Press, San Diego). Also provided are polypeptides, antibodies,and methods of using them, including using nucleic acid expressionmethods. Methods for modulating or intervening in the control of agrowth factor dependent physiology or an immune response are provided.

The present invention is based, in part, upon the discovery of a newcytokine sequence exhibiting significant sequence and structuralsimilarity to G-CSF and IL-6. In particular, it provides primate, e.g.,human, and rodent, e.g., mouse, genes encoding a protein whose maturesize is about 198 amino acids. Functional equivalents exhibitingsignificant sequence homology will be available from other mammalian,e.g., cow, horse, and rat species.

Moreover, the present invention identifies a second associated componentof a complex. Compositions related to the combination of components inthe complex are provided, along with methods of use.

In one embodiment, the invention provides a substantially pure orrecombinant polypeptide comprising the mature protein portion of SEQ IDNO: 2 or 4. Preferably, the polypeptide is: detectably labeled;unglycosylated; denatured; attached to a solid substrate; conjugated toanother chemical moiety; or in a sterile composition. Kit forms includethose comprising the polypeptide and: a compartment comprising thepolypeptide; or with instructions for use or disposal of reagents in thekit.

Binding compounds include those comprising an antigen binding site froman antibody that specifically binds to the described polypeptide. Thebinding compound can also be in a kit comprising: a-compartmentcomprising the binding compound; or with instructions for use ordisposal of reagents in the kit.

The invention further provides a method of producing an antigen:antibodycomplex, comprising contacting, under appropriate conditions, a primateIL-B60 polypeptide with an antibody that specifically or selectivelybinds the polypeptide of the invention, thereby allowing the complex toform.

Nucleic acid embodiments include an isolated or recombinantpolynucleotide encoding the mature protein portion of SEQ ID NO: 2 or 4.

In other embodiments, the invention provides an isolated soluble complexcomprising the mature protein portion of SEQ ID NO: 2 or 4, and themature protein portion of SEQ ID NO: 12 or 13. Preferably the complex:comprises a recombinant polypeptide of SEQ ID NO: 2, 4, 12, or 13; isdetectably labeled; is in a buffered solution; is in a sterile solution.Kits are provided containing such a complex and: a compartmentcomprising the complex; or instructions for use or disposal of reagentsin the kit.

Binding compounds are provided comprising an antigen binding site froman antibody that specifically binds to the soluble complex but not tothe mature polypeptide of SEQ ID NO: 12 or 13. Kits are providedcomprising the binding compound and: a compartment comprising thebinding compound; or instructions for use or disposal of reagents in thekit.

Methods are provided, e.g., of producing an antigen:antibody complex,comprising contacting, under appropriate conditions, a primate complexcomprising IL-B60 and CLF-1 polypeptides with an antibody thatselectively or specifically binds to an isolated soluble complexcomprising the mature protein portion of SEQ ID NO: 2 or 4, and themature protein portion of SEQ ID NO: 12 or 13, thereby allowing thecomplex to form.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding the mature protein portion of SEQ ID NO: 2 or 4, and the matureprotein portion of SEQ ID NO: 12 or 13.

The invention also provides a composition of matter selected from: anisolated polypeptide comprising at least seven amino acids identical tosegments of SEQ ID NO: 2 or 4; a substantially pure or recombinantpolypeptide comprising at least two distinct nonoverlapping segments ofat least five amino acids identical to segments of SEQ ID NO: 2 or 4; anatural sequence polypeptide comprising mature SEQ ID NO: 2 or 4; or afusion polypeptide comprising IL-B60 sequence. In certain embodiments,the distinct nonoverlapping segments of identity include: one of atleast eight amino acids; one of at least five amino acids and a secondof at least six amino acids; at least three segments of at least four,five, and six amino acids, or one of at least twelve amino acids. Inother embodiments the polypeptide of the composition of matter: is thepolypeptide which: comprises a mature sequence of Table 1; is anunglycosylated form of IL-B60; is from a primate, such as a human;comprises at least seventeen amino acids of SEQ ID NO: 2 or 4; exhibitsat least four nonoverlapping segments of at least seven amino acids ofSEQ ID NO: 2 or 4; is a natural allelic variant of IL-B60; has a lengthat least about 30 amino acids; exhibits at least two non-overlappingepitopes which are specific for a primate IL-B60; is glycosylated; has amolecular weight of at least 30 kD with natural glycosylation; is asynthetic polypeptide; is attached to a solid substrate; is conjugatedto another chemical moiety; is a 5-fold or less substitution fromnatural sequence; is a deletion or insertion variant from a naturalsequence; or which further comprises: at least seven amino acidsidentical to segments of SEQ ID NO: 12 or 13; at least two distinctnonoverlapping segments of at least five amino acids identical tosegments of SEQ ID NO: 12 or 13; a natural sequence polypeptidecomprising mature SEQ ID NO: 12 or 13; or a primate CLF-1. In additionalpreferred embodiments, the composition comprises: a substantially pureIL-B60 and CLF-1; a sterile IL-B60 polypeptide comprising the matureprotein of SEQ ID NO: 2 or 4; or the described polypeptide and acarrier, wherein the carrier is: an aqueous compound, including water,saline, and/or buffer; and/or formulated for oral, rectal, nasal,topical,mRNA,or parenteral administration. The invention provides fusionpolypeptides which comprise: mature protein sequence of Table 1; adetection or purification tag, including a FLAG, His6, or Ig sequence;or sequence of another cytokine receptor family protein, includingCLF-1. Kit embodiments include those comprising the polypeptide of thecomposition and: a compartment comprising the protein or polypeptide; orinstructions for use or disposal of reagents in the kit.

The invention further provides methods of using the describedpolypeptides: to label the polypeptide, comprising labeling thepolypeptide with a radioactive label; to separate the polypeptide fromanother polypeptide in a mixture, comprising running the mixture on achromatography matrix, thereby separating the polypeptides; to identifya compound that binds selectively to the polypeptide, comprisingincubating the compound with the polypeptide under appropriateconditions; thereby causing the component to bind to the polypeptide; orto conjugate the polypeptide to a matrix, comprising derivatizing thepolypeptide with a reactive reagent, and conjugating the polypeptide tothe matrix.

Related binding compounds include those comprising an antigen bindingsite from an antibody that specifically or selectively binds to anatural polypeptide, as described above, wherein: the binding compoundis in a container; the IL-B60 polypeptide is from a human; the bindingcompound is an Fv, Fab, or Fab2 fragment; the binding compound isconjugated to another chemical moiety; or the antibody: is raisedagainst a mature polypeptide of Table 1; is raised against a matureIL-B60; is raised to a purified human IL-B60; is immunoselected; is apolyclonal antibody; binds to a denatured IL-B60; exhibits a Kd toantigen of at least 30 μM; is attached to a solid substrate, including abead or plastic membrane; is in a sterile composition; or is detectablylabeled, including a radioactive or fluorescent label. Kits are providedcomprising such a binding compound and: a compartment comprising thebinding compound; or instructions for use or disposal of the reagents ofthe kit.

Methods are provided for producing an antigen:antibody complex,comprising contacting, under appropriate conditions, a primate IL-B60polypeptide with a described antibody, thereby allowing the complex toform. Preferably, in the method: the complex is purified from othercytokines; the complex is purified from other antibody; the contactingis with a sample comprising a cytokine; the contacting allowsquantitative detection of the antigen; the contacting is with a samplecomprising the antibody; or the contacting allows quantitative detectionof the antibody.

In another embodiment the invention includes a composition comprising: asterile binding compound, as described, or the binding compound and acarrier, wherein the carrier: wherein the carrier is: an aqueouscompound, including water, saline, and/or buffer; and/or formulated fororal, rectal, nasal, topical, or parenteral administration.

Nucleic acid embodiments include an isolated or recombinant nucleic acidencoding the described polypeptide, wherein: the IL-B60 is from a human;or the nucleic acid: encodes an antigenic peptide sequence of Table 1;encodes a plurality of antigenic peptide sequences of Table 1; encodes aplurality of antigenic peptide sequences of Table 4; exhibits identityover at least thirteen nucleotides to a natural cDNA encoding thesegment; is an expression vector; further comprises an origin ofreplication; is from a natural source; comprises a detectable label;comprises synthetic nucleotide sequence; is less than 6 kb, preferablyless than 3 kb; is from a primate; comprises a natural full lengthcoding sequence; is a hybridization probe for a gene encoding theIL-B60; or is a PCR primer, PCR product, or mutagenesis primer.Preferred embodiments include where the isolated or recombinant nucleicacid is in a cell or tissue. The cell may be: a prokaryotic cell; aeukaryotic cell; a bacterial cell; a yeast cell; an insect cell; amammalian cell; a mouse cell; a primate cell; or a human cell.

Kits are provided comprising the described nucleic acid and: acompartment comprising the nucleic acid; a compartment furthercomprising a primate IL-B60 polypeptide; or instructions for use ordisposal of reagents in the kit.

The invention further provides methods for forming a duplex with apolynucleotide described above, comprising contacting the polynucleotidewith a probe that hybridizes, under stringent conditions, to at least 25contiguous nucleotides of the coding portion of SEQ ID NO: 1, 3, orencoding the nature SEQ ID NO: 12 or 13; thereby forming the duplex.

In a further aspect, the invention provides a nucleic acid which:hybridizes under wash conditions of 30 minutes at 30° C. and less than2M salt to the coding portion of SEQ ID NO: 1; or exhibits identityover-a stretch of at least about 30 nucleotides to a primate IL-B60. Inpreferred embodiments, the wash conditions that are at 45° C. and/or 500mM salt; or at 55° C. and/or 150 mM salt; or the stretch is at least 55nucleotides, e.g., at least 75 nucleotides.

Methods are provided, e.g., of modulating physiology or development of acell or tissue culture cells comprising contacting the cell with anagonist or antagonist of a mammalian IL-B60; or contacting the cell withan agonist or antagonist of a complex comprising mammalian IL-B60 andCLF-1. Additionally, the invention provides a method of increasing thesecretion of: an IL-B60 sequence, comprising expressing the polypeptidewith CLF-1; or a CLF-1, comprising expressing the CLF-1 with an IL-B60sequence. In preferred embodiments of the method, the increasing is atleast 3 fold, 5×, 7×, 10×, or more; or the expressing is of arecombinant nucleic acid encoding one or both of the polypeptide andCLF-1.

The invention further provides a method of screening for a receptorwhich binds an isolated soluble complex comprising the mature proteinportion of SEQ ID NO: 2 or 4, and the mature protein portion of SEQ IDNO: 12 or 13, comprising contacting the complex to a cell expressing thereceptor under conditions allowing the complex to bind to the receptor,thereby forming a detectable interaction. Preferably, the interactionresults in a physiological response in the cell.

Other embodiments of the invention include, e.g., an isolated solublecomplex comprising at least 6 amino acids of the mature protein portionof SEQ ID NO: 2 or 4, and: at least 6 amino acids of the mature proteinportion of SEQ ID NO: 12 or 13; or at least 6 amino acids of the matureprotein portion of the CNTF-R. Such complex may, e.g., comprise arecombinant polypeptide of mature SEQ ID NO: 2 or 4; comprise arecombinant polypeptide of mature SEQ ID NO: 12 or 13; comprise arecombinant polypeptide of mature CNTF-R; comprise both a recombinantpolypeptide of mature SEQ ID NO: 2 or 4, and a recombinant polypeptideof mature SEQ ID NO: 12 or 13; comprise both a recombinant polypeptideof mature SEQ ID NO: 2 or 4, and a recombinant polypeptide of matureCNTF-R; be detectably labeled; be in a buffered solution; or be in asterile solution. Preferred embodiments include those which: comprise amature IL-B60 polypeptide; comprise a mature CLF-1 polypeptide;comprises a mature CNTF-R polypeptide; exhibit at least fournonoverlapping segments of at least seven amino acids of SEQ ID NO: 2 or4; exhibit epitopes from both primate L-B60 and primate CLF-1; exhibitepitopes from both primate L-B60 and primate CNTF-R; not beglycosylated; be attached to a solid substrate; be conjugated to anotherchemical moiety; or comprise a detection or purification tag, includinga FLAG, His6, or Ig sequence.

Kits are provided, e.g., comprising the complex and: a compartmentcomprising the complex, and/or instructions for use or disposal ofreagents in the kit.

Fusion polypeptides are provided, which include, e.g., an isolated orrecombinant polypeptide comprising: a first segment comprising at leastseven amino acids identical to segments of SEQ ID NO: 2 or 4, and asecond segment comprising at least seven amino acids identical tosegments of mature SEQ ID NO: 12 or 13; at least two distinctnonoverlapping segments of at least five amino acids identical tosegments of mature SEQ ID NO: 2 or 4, and a third segment comprising atleast seven amino acids identical to segments of mature SEQ ID NO: 12 or13; at least one segment comprising at least seven amino acids identicalto segments of mature SEQ ID NO: 2 or 4, and two distinct nonoverlappingsegments of at least five amino acids identical to segments of matureSEQ ID NO: 12 or 13; a first segment comprising at least seven aminoacids identical to segments of SEQ ID NO: 2 or 4, and a second segmentcomprising at least seven amino acids identical to segments of matureprimate CNTF-R; at least two distinct nonoverlapping segments of atleast five amino acids identical to segments of mature SEQ ID NO: 2 or4, and a third segment comprising at least seven amino acids identicalto segments of mature primate CNTF-R; or at least one segment comprisingat least seven amino acids identical to segments of mature SEQ ID NO: 2or 4, and two distinct nonoverlapping segments of at least five aminoacids identical to segments of mature primate CNTF-R. Certainembodiments include those wherein the distinct nonoverlapping segmentsof identity: include one of at least eight amino acids; include one ofat least five amino acids and a second of at least six amino acids;include at least three segments of at least four, five, and six aminoacids, or include one of at least twelve amino acids. Other embodimentsinclude those which: comprise a mature IL-B60 sequence; comprise amature CLF-1 sequence; comprise a mature CNTF-R sequence; exhibit atleast four nonoverlapping segments of at least seven amino acids of SEQID NO: 2 or 4; have a length at least about 30 amino acids; exhibitepitopes from both primate IL-B60 and primate CLF-1; exhibits epitopesfrom both primate IL-B60 and primate CNTF-R; are not glycosylated; havea molecular weight of at least 30 kD; be a synthetic polypeptide; beattached to a solid substrate; be conjugated to another chemical moiety;or comprise a detection or purification tag, including a FLAG, His6, orIg sequence.

Other embodiments include a composition comprising: substantially purecombination of IL-B60 and CLF-l; substantially pure combination ofIL-B60 and CNTF-R; a sterile polypeptide described above; or thepolypeptide described above and a carrier, wherein the carrier is: anaqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration. A kit is provided comprising a polypeptide as described,and: a compartment comprising the polypeptide; and/or instructions foruse or disposal of reagents in the kit.

Methods are also provided, e.g., of making an antibody which recognizesa complex as described, comprising inducing an immune response in ananimal with the complex; of immunoselecting antibodies, comprisingcontacting a population of antibodies to a complex as described, andseparating antibodies that bind from those which do not bind; or offormulating a composition, comprising admixing a complex as describedwith a carrier.

Binding compounds are provided, e.g., comprising an antigen binding sitefrom an antibody, which antibody specifically binds a described complex,but not to any of the mature polypeptides of SEQ ID NO: 2, 4, 12, 13, orCNTF-R. Certain embodiments include those wherein: the binding compoundis: in a container; an Fv, Fab, or Fab2 fragment; or conjugated toanother chemical moiety; or the antibody: is raised against asubstantially pure complex of IL-B60 with CLF-1; is raised against asubstantially pure complex of IL-B60 with CNTF-R; is immunoselected; isa polyclonal antibody; exhibits a Kd to antigen of at least 30 μM; isattached to a solid substrate, including a bead or plastic membrane; isin a sterile composition; or is detectably labeled, including aradioactive or fluorescent label. Additional embodiments include acomposition comprising: a sterile binding compound as described, or thebinding compound as described and a carrier, wherein the carrier is: anaqueous compound, including water, saline, and/or buffer; and/orformulated for oral, rectal, nasal, topical, or parenteraladministration.

With the binding composition are provided a kit comprising the bindingcompound and: a compartment comprising the binding compound; orinstructions for use or disposal of reagents in the kit. Also providedare methods of producing an antigen:antibody complex, comprisingcontacting under appropriate conditions a primate complex comprising:IL-B60 and CLF-1 polypeptides; or IL-B60 and CNTF-R polypeptides; withan antibody as described, thereby allowing the complex to form.Preferably, in the method, the complex is purified from other cytokines;the complex is purified from other antibody; the contacting is with asample comprising a cytokine; the contacting allows quantitativedetection of the antigen; the contacting is with a sample comprising theantibody; or the contacting allows quantitative detection of theantibody.

Various nucleic acids are provided, e.g., an isolated or recombinantnucleic acid: encoding the amino acid portions described above; encodingthe amino acid portions as described, and comprise a segment at least 30contiguous nucleotides from SEQ ID NO: 1 or 3; which will coexpress asegment of at least seven contiguous amino acids from SEQ ID NO: 2 or 4,and a segment of at least seven contiguous amino acids from SEQ ID NO:12 or 13; or which will coexpress a segment of at least seven contiguousamino acids from SEQ ID NO: 2 or 4, and a segment of at least sevencontiguous amino acids from CNTF-R. Preferred nucleic acids includethose which, e.g.,: encode IL-B60 from a human; encode CLF-1 from ahuman; encodes CNTF-R from a human; are an expression vector; furthercomprise an origin of replication; comprise a detectable label; comprisesynthetic nucleotide sequence; or are less than 6 kb, preferably lessthan 3 kb. A cell comprising the recombinant nucleic acid is provided,e.g., wherein the cell is: a prokaryotic cell; a eukaryotic cell; abacterial cell; a yeast cell; an insect cell; a mammalian cell; a mousecell; a primate cell; or a human cell. Various nucleic acid kits areprovided, e.g., comprising the nucleic acid and: a compartmentcomprising the nucleic acid; a compartment further comprising a primateIL-B60 polypeptide; a compartment further comprising a primate CLF-1polypeptide; a compartment further comprising a primate CNTF-Rpolypeptide; or instructions for use or disposal of reagents in the kit.Methods are also provided, e.g., of making a duplex nucleic acid,comprising contacting such a nucleic acid with a complementary nucleicacid under appropriate conditions, thereby forming said duplex; ofexpressing a polypeptide, comprising expressing the nucleic acid,thereby producing the polypeptide; or of transfecting a cell, comprisingcontacting said cell under appropriate conditions with the nucleic acid,thereby transfecting the cell.

In an alternative embodiment, the invention provides an isolated orrecombinant nucleic acid which encodes at least 5 contiguous amino acidsof SEQ ID NO: 12, 13, or primate CNTF-R and: hybridizes under washconditions of 30 minutes at 30° C. and less than 2M salt to the codingportion of SEQ ID NO: 1; or exhibits identity over a stretch of at leastabout 30 nucleotides to a primate IL-B60. Preferred embodiments include:the isolated nucleic acid, wherein: the contiguous amino acids number atleast 8; the wash conditions are at 45° C. and/or 500 mM salt; or thestretch is at least 55 nucleotides; or the recombinant nucleic acid,wherein: the contiguous amino acids number at least 12; the washconditions are at 55° C. and/or 150 mM salt; or the stretch is at least75 nucleotides.

The invention particularly provides methods of modulating physiology ordevelopment of a cell or tissue culture cells comprising contacting thecell with an agonist or antagonist of a complex comprising mammalianIL-B60 and: CLF-1; or CNTF-R. It also provides methods of producing theproteins, e.g., producing a complex described, comprising coexpressing arecombinant IL-B60 with a recombinant CLF-1 or CNTF-R; increasing thesecretion of an IL-B60 polypeptide comprising expressing the polypeptidewith CLF-1 or CNTF-R; or increasing the secretion of a CLF-1polypeptide, comprising expressing the CLF-1 with an IL-B60. Typically,the increasing is at least 3 fold; or the expressing is of a recombinantnucleic acid encoding one or both of the polypeptide and CLF-1.

Also provided are methods of screening for a receptor which binds thedescribed complex, comprising contacting the complex to a cellexpressing the receptor under conditions allowing the complex to bind tothe receptor, thereby forming a detectable interaction. Preferably, theinteraction results in a physiological response in the cell.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by reference.

Outline

I. General II. Purified IL-B60 or complex A. physical properties B.biological properties III. Physical Variants A. sequence variants,fragments B. post-translational variants 1.  glycosylation 2.  othersIV. Functional Variants A. analogs, fragments 1.  agonists2.  antagonists B. mimetics 1.  protein 2.  chemicals C. speciesvariants V. Antibodies A. polyclonal B. monoclonal C. fragments, bindingcompositions VI. Nucleic Acids A. natural isolates; methods B. syntheticgenes C. methods to isolate VII. Making IL-B60 or complex, mimetics A.recombinant methods B. synthetic methods C. natural purification VIII.Uses A. diagnostic B. therapeutic IX. Kits A. nucleic acid reagents B.protein reagents C. antibody reagents X. Isolating receptors for IL-B60or complexI. General

The present invention provides amino acid sequences and DNA sequencesencoding various mammalian proteins which are cytokines, e.g., which aresecreted molecules which can mediate a signal between immune or othercells. See, e.g., Paul (1998) Fundamental Immunology (4th ed.) RavenPress, N.Y. The full length cytokines, and fragments, or antagonistswill be useful, e.g., in physiological modulation of cells expressing areceptor. It is likely that IL-B60 has either stimulatory or inhibitoryeffects on hematopoietic cells, including, e.g., lymphoid cells, such asT-cells, B-cells, natural killer (NK) cells, macrophages, dendriticcells, hematopoietic progenitors, etc. The proteins will also be usefulas antigens, e.g., immunogens, for raising antibodies to variousepitopes on the protein, both linear and conformational epitopes.

A sequence encoding IL-B60 was identified from a human genomic sequence.The molecule was designated huIL-B60. A rodent sequence, e.g., frommouse, is also described.

The human gene encodes a small soluble cytokine-like protein, of about198 amino acids. The psort predicted signal sequence probably is about17 residues, and would run from the Met to about Ala. See Table 1 andSEQ. ID. NO: 1 and 2. IL-B60 exhibits structural motifs characteristicof a member of the long chain cytokines. Compare, e.g., IL-B60, G-CSF,and IL-6, sequences available from GenBank. Closest matching is withCT-1, oncostatin M, and CNTF. See also Table 2.

TABLE 1 Nucleic acid (SEQ ID NO: 1) encoding IL-B60 from a primate,e.g., human. Predicted signal cleavage site is indicated. Nucleotide 375may be A. Translated amino acid sequence is SEQ ID NO: 2. ccgagcgaaaaaaacctgcg agtgggcctg gcggatggga ttattaaagc ttcgccggag   60 ccgcggctcgccctcccact ccgccagcct ccgggagagg agccgcaccc ggccggcccg  120 gccccagccccatggacctc cgagcagggg actcgtgggg g atg tta gcg tgc ctg  176                                              Met Leu Ala Cys Leu                                                      −15 tgc acg gtgctc tgg cac ctc cct gca gtg cca gct ctc aat cgc aca  224 Cys Thr Val LeuTrp His Leu Pro Ala Val Pro Ala Leu Asn Arg Thr        −10                  −5              −1   1 ggg gac cca ggg cctggc ccc tcc atc cag aaa acc tat gac ctc acc  272 Gly Asp Pro Gly Pro GlyPro Ser Ile Gln Lys Thr Tyr Asp Leu Thr  5                  10                  15                  20 cgc tacctg gag cac caa ctc cgc agc ttg gct ggg acc tat ctg aac  320 Arg Tyr LeuGlu His Gln Leu Arg Ser Leu Ala Gly Thr Tyr Leu Asn                 25                  30                  35 tac ctg ggcccc cct ttc aac gag cca gac ttc aac cct ccc cgc ctg  368 Tyr Leu Gly ProPro Phe Asn Glu Pro Asp Phe Asn Pro Pro Arg Leu             40                  45                  50 ggg gca gag actctg ccc agg gcc act gtt gac ttg gag gtg tgg cga  416 Gly Ala Glu Thr LeuPro Arg Ala Thr Val Asp Leu Glu Val Trp Arg         55                  60                  65 agc ctc aat gac aaactg cgg ctg acc cag aac tac gag gcc tac agc  464 Ser Leu Asn Asp Lys LeuArg Leu Thr Gln Asn Tyr Glu Ala Tyr Ser     70                  75                  80 cac ctt ctg tgt tac ttgcgt ggc ctc aac cgt cag gct gcc act gct  512 His Leu Leu Cys Tyr Leu ArgGly Leu Asn Arg Gln Ala Ala Thr Ala 85                  90                  95                 100 gag ctgcgc cgc agc ctg gcc cac ttc tgc acc agc ctc cag ggc ctg  560 Glu Leu ArgArg Ser Leu Ala His Phe Cys Thr Ser Leu Gln Gly Leu                105                 110                 115 ctg ggc agcatt gcg ggc gtc atg gca gct ctg ggc tac cca ctg ccc  608 Leu Gly Ser IleAla Gly Val Met Ala Ala Leu Gly Tyr Pro Leu Pro            120                 125                 130 cag ccg ctg cctggg act gaa ccc act tgg act cct ggc cct gcc cac  656 Gln Pro Leu Pro GlyThr Glu Pro Thr Trp Thr Pro Gly Pro Ala His        135                 140                 145 agt gac ttc ctc cagaag atg gac gac ttc tgg ctg ctg aag gag ctg  704 Ser Asp Phe Leu Gln LysMet Asp Asp Phe Trp Leu Leu Lys Glu Leu    150                 155                 160 cag acc tgg ctg tgg cgctcg gcc aag gac ttc aac cgg ctc aag aag  752 Gln Thr Trp Leu Trp Arg SerAla Lys Asp Phe Asn Arg Leu Lys Lys165                 170                 175                 180 aag atgcag cct cca gca gct gca gtc acc ctg cac ctg ggg gct cat  800 Lys Met GlnPro Pro Ala Ala Ala Val Thr Leu His Leu Gly Ala His                185                 190                 195 ggc ttctgacttctga ccttctcctc ttcgctcccc cttcaaaccc tgctcccact  856 Gly Phettgtgagagc cagccctgta tgccaacacc tgttgagcca ggagacagaa gctgtgagcc  916tctggccctt tcctggaccg gctgggcgtg tgatgcgatc agccctgtct cctccccacc  976tcccaaaggt ctaccgagct ggggaggagg tacagtaggc cctgtcctgt cctgtttcta 1036caggaagtca tgctcgaggg agtgtgaagt ggttcaggtt ggtgcagagg cgctcatggc 1096ctcctgcttc ttgcctacca cttggccagt gcccacccag cccctcaggt ggcacatctg 1156gagggcaggg gttgaggggc caccaccaca catgcctttc tggggtgaag ccctttggct 1216gccccactct ccttggatgg gtgttgctcc cttatcccca aatcactcta tacatccaat 1276tcaggaaaca aacatggtgg caattctaca caaaaagaga tgagattaac agtgcagggt 1336tggggtctgc attggaggtg ccctataaac cagaagagaa aatactgaaa gcacaggggc 1396agggacagac cagaccagac ccaggagtct ccaaagcaca gagtggcaaa caaaacccga 1456gctgagcatc aggaccttgc ctcgaattgt cttccagtat tacggtgcct cttctctgcc 1516ccctttccca gggtatctgt gggttgccag gctggggagg gcaaccatag ccacaccaca 1576ggatttcctg aaagtttaca atgcagtagc attttggggt gtagggtggc agctccccaa 1636ggccctgccc cccagcccca cccactcatg actctaagtg tgttgtatta atatttattt 1696atttggagat gttatttatt agatgatatt tattgcagaa tttctattct tgtattaaca 1756aataaaatgc ttgccccaga acaaaaaaaa aaaa 1790MLACLCTVLWHLPAVPA/LNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVDLEVWRSLNDKLRLTQNYEAYSHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMAALGYPLPQPLPGTEPTWTPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAAAVTLHLGAHGF Rodent, e.g., mouse, IL-B60 (SEQ ID NO: 3 and 4): atg tta gcttgc cta tgc acg gtg ctg tgg cac ctc cct gca gtg cca   48 Met Leu Ala CysLeu Cys Thr Val Leu Trp His Leu Pro Ala Val Pro        −15                 −10                  −5 gct ctt aat cgc acagga gat cca ggc cct ggc ccc tcc atc cag aaa   96 Ala Leu Asn Arg Thr GlyAsp Pro Gly Pro Gly Pro Ser Ile Gln Lys -1   1               5                  10                  15 acc tatgac ctc acc cgc tac ctg gag cat caa ctc cgc agc tta gct  144 Thr Tyr AspLeu Thr Arg Tyr Leu Glu His Gln Leu Arg Ser Leu Ala                 20                  25                  30 ggg acc tacctg aac tac ctg ggg ccc cct ttc aac gag cct gac ttc  192 Gly Thr Tyr LeuAsn Tyr Leu Gly Pro Pro Phe Asn Glu Pro Asp Phe             35                  40                  45 aat cct cct cgactg ggg gca gaa act ctg ccc agg gcc acg gtc aac  240 Asn Pro Pro Arg LeuGly Ala Glu Thr Leu Pro Arg Ala Thr Val Asn         50                  55                  60 ttg gaa gtg tgg cgaagc ctc aat gac agg ctg cgg ctg acc cag aac  288 Leu Glu Val Trp Arg SerLeu Asn Asp Arg Leu Arg Leu Thr Gln Asn     65                  70                  75 tat gag gcg tac agt cacctc ctg tgt tac ttg cgt ggc ctc aac cgt  336 Tyr Glu Ala Tyr Ser His LeuLeu Cys Tyr Leu Arg Gly Leu Asn Arg 80                  85                  90                  95 cag gctgcc aca gct gaa ctc cga cgt agc ctg gcc cac ttc tgt acc  384 Gln Ala AlaThr Ala Glu Leu Arg Arg Ser Leu Ala His Phe Cys Thr                100                 105                 110 agc ctc cagggc ctg ctg ggc agc att gca ggt gtc atg gcg acg ctt  432 Ser Leu Gln GlyLeu Leu Gly Ser Ile Ala Gly Val Met Ala Thr Leu            115                 120                 125 ggc tac cca ctgccc cag cct ctg cca ggg act gag cca gcc tgg gcc  480 Gly Tyr Pro Leu ProGln Pro Leu Pro Gly Thr Glu Pro Ala Trp Ala        130                 135                 140 cct ggc cct gcc cacagt gac ttc ctc cag aag atg gat gac ttc tgg  528 Pro Gly Pro Ala His SerAsp Phe Leu Gln Lys Met Asp Asp Phe Trp    145                 150                 155 ctg ctg aag gag ctg cagacc tgg cta tgg cgt tca gcc aag gac ttc  576 Leu Leu Lys Glu Leu Gln ThrTrp Leu Trp Arg Ser Ala Lys Asp Phe160                 165                 170                 175 aac cggctt aag aag aag atg cag cct cca gca gct tca gtc acc ctg  624 Asn Arg LeuLys Lys Lys Met Gln Pro Pro Ala Ala Ser Val Thr Leu                180                 185                 190 cac ttg gaggcc cat ggt ttc tga  648 His Leu Glu Ala His Gly Phe             195MLACLCTVLWHLPAVPA/LNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVNLEVWRSLNDRLRLTQNYEAYSHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMATLGYPLPQPLPGTEPAWAPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAASVTLHLEAHGF

TABLE 2 Comparison of primate and rodent embodiments of IL-B60, both thenucleotide and amino acid sequences. hIL-B60ATGTTAGCGTGCCTGTGCACGGTGCTCTGGCACCTCCCTGCAGTGCCAGCTCTCAATCGC mIL-B60ATGTTAGCTTGCCTATGCACGGTGCTGTGGCACCTCCCTGCAGTGCCAGCTCTTAATCGC ************* *********** ************************** ****** hIL-B60ACAGGGGACCCAGGGCCTGGCCCCTCCATCCAGAAAACCTATGACCTCACCCGCTACCTG mIL-B60ACAGGAGATCCAGGCCCTGGCCCCTCCATCCAGAAAACCTATGACCTCACCCGCTACCTG ***** ******* ********************************************* hIL-B60GAGCACCAACTCCGCAGCTTGGCTGGGACCTATCTGAACTACCTGGGCCCCCCTTTCAAC mIL-B60GAGCATCAACTCCGCAGCTTAGCTGGGACCTACCTGAACTACCTGGGGCCCCCTTTCAAC ******************* *********** ************** ************ hIL-B60GAGCCAGACTTCAACCCTCCCCGCCTGGGGGCAGAGACTCTGCCCAGGGCCACTGTTGAC mIL-B60GAGCCTGACTTCAATCCTCCTCGACTGGGGGCAGAAACTCTGCCCAGGGCCACGGTCAAC ************* ***** ** *********** ***************** **  ** hIL-B60TTGGAGGTGTGGCGAAGCCTCAATGACAAACTGCGGCTGACCCAGAACTACGAGGCCTAC mIL-B60TTGGAAGTGTGGCGAAGCCTCAATGACAGGCTGCGGCTGACCCAGAACTATGAGGCGTAC ***************************  ******************** ***** *** hIL-B60AGCCACCTTCTGTGTTACTTGCGTGGCCTCAACCGTCAGGCTGCCACTGCTGAGCTGCGC mIL-B60AGTCACCTCCTGTGTTACTTGCGTGGCCTCAACCGTCAGGCTGCCACAGCTGAACTCCGA ** ******************************************* ***** ** ** hIL-B60CGCAGCCTGGCCCACTTCTGCACCAGCCTCCAGGGCCTGCTGGGCAGCATTGCGGGCGTC mIL-B60CGTAGCCTGGCCCACTTCTGTACCAGCCTCCAGGGCCTGCTGGGCAGCATTGCAGGTGTC ******************* ******************************** ** *** hIL-B60ATGGCAGCTCTGGGCTACCCACTGCCCCAGCCGCTGCCTGGGACTGAACCCACTTGGACT mIL-B60ATGGCGACGCTTGGCTACCCACTGCCCCAGCCTCTGCCAGGGACTGAGCCAGCCTGGGCC *****  * ********************** ***** ******** **  * *** * hIL-B60CCTGGCCCTGCCCACAGTGACTTCCTCCAGAAGATGGACGACTTCTGGCTGCTGAAGGAG mIL-B60CCTGGCCCTGCCCACAGTGACTTCCTCCAGAAGATGGATGACTTCTGGCTGCTGAAGGAG************************************** ********************* hIL-B60CTGCAGACCTGGCTGTGGCGCTCGGCCAAGGACTTCAACCGGCTCAAGAAGAAGATGCAG mIL-B60CTGCAGACCTGGCTATGGCGTTCAGCCAAGGACTTCAACCGGCTTAAGAAGAAGATGCAG************** ***** ** ******************** *************** hIL-B60CCTCCAGCAGCTGCAGTCACCCTGCACCTGGGGGCTCATGGCTTCTGA mIL-B60CCTCCAGCAGCTTCAGTCACCCTGCACTTGGAGGCCCATGGTTTCTGA ************************** *** *** ***** ****** Alignment of IL-B60: underlined areproposed helices. In general, those residues that are in helix A and Dand not pointing inward toward the core (mostly the hydrophobic residuesin A and D helix) are the most likely residues to interact withreceptors.                                       A hIL-B60MLACLCTVLWHLPAVPALNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGT mIL-B60MLACLCTVLWHLPAVPALNRTGDPGPGPSIQKTYDLTRYLEHQLRSLAGT**************************************************                                            B hIL-B60YLNYLGPPFNEPDFNPPRLGAETLPRATVDLEVWRSLNDKLRLTQNYEAY mIL-B60YLNYLGPPFNEPDFNPPRLGAETLPRATVNLEVWRSLNDRLRLTQNYEAY*****************************:*********:**********                                      C hIL-B60SHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMAALGYPLPQ mIL-B60SHLLCYLRGLNRQAATAELRRSLAHFCTSLQGLLGSIAGVMATLGYPLPQ******************************************:*******                                      D hIL-B60PLPGTEPTWTPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQ mIL-B60PLPGTEPAWAPGPAHSDFLQKMDDFWLLKELQTWLWRSAKDFNRLKKKMQ*******:*:**************************************** hIL-B60PPAAAVTLHLGAHGF mIL-B60 PPAASVTLHLEAHGF ****:***** ****

TABLE 3 Comparison of various cytokines compared to IL-B60. Human IL-B60is SEQ ID NO: 2; mouse IL-B60 is SEQ ID NO: 4; mouse LIF (mLIF) is SEQID NO: 5 and Accession number X06381; human LIF (hLIF) is SEQ ID NO: 6and Accession numbers M63420 J05436; human CT-1 (hCT-1) is SEQ ID NO: 7and Accession number U43030; mouse CT-1 (mCT-1) is SEQ ID NO: 8 andAccession number U18366; human CNTF (hCNTF) is SEQ ID NO: 9 andAccession number X60542; mouse CNTF (mCNTF) is SEQ ID NO: 10 andAccession number U05342; human DNAX IL-40 (hDIL-40) is SEQ ID NO: 11.mLIF -MKVLAAGIVPLLLLVLHWKHGAGSPLPI-TPVNATC-AIRHPCHGNLMN hLIF-MKVLAAGVVP-LLLVLHWKHGAGSPLPI-TPVNATC-AIRHPCHNNLMN hCT-1--MSRREGSLE---D--PQTDSSVSLLPH-LEA-----KIRQT-HS--LA mCT-1--MSQREGSLE---D--HQTDSSISFLPH-LEA-----KIRQT-HN--LA hIL-B60-MLACLCTVLW------HLPAVPALNRTG-DPG-PGP-SIQKT-YD--LT mIL-B60-MLACLCTVLW------HLPAVPALNRTG-DPG-PGP-SIQKT-YD--LT hCNTF------MAFTE------HSPLTPHR-R---D-L-CSR-SIW-------LA mCNTF------MAFAE------QSPLTLHR-R---D-L-CSR-SIW-------LA hDIL-40MTHLSLLGPLPCVRTSQQLPETQQVTTPGKKPVSVGRREVRVP-----GT                                       :     . mLIFQIKNQLAQLNQSANALFISYYTAQGEPF--PNNVEK-LCAPNMTDFPSFH hLIFQIRSQLAQLNGSANALFILYYTAQGEPF--PNNLDK-LCGPNVTDFPPFH hCT-1HLLTKYAEQ------LLQEYVQLQGDPFGLPSFSPPRLPVAGLSAPAPSH mCT-1RLLTKYAEQ------LLEEYVQQQGEPFGLPGFSPPRLPLAGLSGPAPSH hIL-B60RYLEHQLRS------LAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVDL mIL-B60RYLEHQLRS------LAGTYLNYLGPPFNEPDFNPPRLGAETLPRATVNL hCNTFRKIRSDLTA------LTESYVKHQG--LNK---NINLDSADGMPVASTD- mCNTFRKIRSDLTA------LMESYVKHQG--LNK---NISLDSVDGVPVASTD- hDIL-40ALVPSLLSV------SVLLQLQYQGSPFSDPGFSAPELQLSSLPPATAFF                        *  :.  .          :.  . mLIF---GNGTEKTKLVELYRMVAYLSASLTNITR-DQKVLNPTAVSLQVKLNA hLIF---ANGTEKAKLVELYRIVVYLGTSLGNITR-DQKILNPSALSLHSKLNA hCT-1---AGLPVHERLRLDAAALAALPPLLDAVCR-RQAELNPRAPRLLRRLED mCT-1---AGLPVSERLRQDAAALSVLPALLDAVRR-RQAELNPRAPRLLRSLED hIL-B60EVWRSLNDKLRLTQNYEAYSHLLCYLRGLN--RQAATAELRRSLAHFCTS mIL-B60EVWRSLNDRLRLTQNYEAYSHLLCYLRGLN--RQAATAELRRSLAHFCTS hCNTF-QWSELTEAERLQENLQAYRTFHVLLARLLEDQQVHFTPTEGDFHQAIHT mCNTF-RWSEMTEAERLQENLQAYRTFQGMLTKLLEDQRVHFTPTEGDFHQAIHT hDIL-40KTWHALDDGERLSLAQRAID---PHLQLVED-DQSDLNPGSPILPAQLGA:         :*             *  :    :         : mLIFTIDVMRGLLSNVLCRLCNKYRV--GHVDVPP-----VPDHSDKE--AFQR hLIFTADILRGLLSNVLCRLCSKYHV--GHVDVTY-----GPDTSGKD--VFQK hCT-1AARQARALGAAVEALLAALGAANRGPRAEPP--AATASAASATG--VFPA mCT-1AARQVRALGAAVETVLAALGAAARGPGPEPVTVATLFTANSTAG--IFSA hIL-B60LQGLLGSIAGVMAALGYPLPQP--LPGTEPT----WTPGPAHS---DFLQ mIL-B60LQGLLGSIAGVMATLGYPLPQP--LPGTEPA----WAPGPAHS---DFLQ hCNTFLLLQVAAFAYQIEELMILLEYK--IPRNEAD----GMPINVGDGG-LFEK mCNTFLTLQVSAFAYQLEELMALLEQK--VPEKEAD----GMPVTIGDGG-LFEK hDIL-40ARLRAQGPLGNMAAIMTALGLP--IP-PEED-----TPGLAAFGASAFER      .    :                         .         * mLIFKKLGCQLLGTYKQVIS----VVVQAF--------------------- hLIFKKLGCQLLGKYKQIIA----VLAQAF--------------------- hCT-1KVLGLRVCGLYREWLSRTEGDLGQLLPGGSA---------------- mCT-1KVLGFHVCGLYGEWVSRTEGDLGQLVPGGVA---------------- hIL-B60KMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAAAVTLHLGAHGF-- mIL-B60KMDDFWLLKELQTWLWRSAKDFNRLKKKMQPPAASVTLHLEAHGF-- hCNTFKLWGLKVLQELSQWTVRSIHDL-RFISSHQTGIPARGSHYIANNKKM mCNTFKLWGLKVLQELSQWTVRSIHDL-RVISSHHMGISAHESHYGA--KQM hDIL-40KCRGYVVTREYGHWTDRAVRDLALLKAKYSA----------------*  .  :        .     .                :   :.

The structural homology of IL-B60 to related cytokine proteins suggestsrelated function of this molecule. IL-B60 is a long chain cytokineexhibiting sequence similarity to IL-6 and G-CSF.

IL-B60 agonists, or antagonists, may also act as functional or receptorantagonists, e.g., which block IL-6 or G-CSF binding to their respectivereceptors, or mediating the opposite actions. Thus, IL-B60, or itsantagonists, may be useful in the treatment of abnormal medicalconditions, including immune disorders, e.g., T cell immunedeficiencies, chronic inflammation, or tissue rejection, or incardiovascular or neurophysiological conditions.

The natural antigens are capable of mediating various biochemicalresponses which lead to biological or physiological responses in targetcells. The preferred embodiment characterized herein is from human, butother primate, or other species counterparts exist in nature. Additionalsequences for proteins in other mammalian species, e.g., primates,canines, felines, and rodents, should also be available. See below. Thedescriptions below are directed, for exemplary purposes, to a humanIL-B60, but are likewise applicable to related embodiments from otherspecies.

In particular, the association of the IL-B60 with a partner has beenconfirmed. The IL-B60 and CLF-1 molecules have likely evolved together,reflected in their homology between species. For example, thecoevolution of their function is suggested by the observation that thehuman/mouse relationship of the IL-B60 is very close, as is thehuman/mouse CLF-1. If the two functionally associate, they might acttogether in the fashion of IL-12. See, e.g., Trinchieri (1998) Adv.Immunol. 70:83-243; Gately, et al.(1998) Ann. Rev. Immunol. 16:495-521;and Trinchieri (1998) Int. Rev. Immunol. 16:365-396.

As a complex, however, they will interact with two tall receptors in thecytokine receptor family, e.g., gp130, LIF-R, OSM-R, IL-12Rb1, IL-12Rb2,and NR30. These receptors will be tested for binding to the solublecomplex. A series of BAF/3 cells that stably express various of thesetall receptors have been constructed.

The supernatants of transfectants of both IL-B60 and CLF-1 (or a singlecombination construct) in the same cell, will be used to test thesevarious BAF/3 cells to see if there is a proliferative or othersignaling response. As such, most of the physiological effects of thecytokine may be due to the complex of the proteins. As such, many of thedescriptions below of biology resulting from the cytokine may actuallybe physiologically effected by the complex comprising the combination ofthe subunits.

Table 4 provides the sequences of the IL-B60 partner, known as CLF-1.The CNTF receptor (CNTF-R) subunit alpha was described, e.g., by Davis,et al. (1991) Science 253:59-63. See also GenBank accession numbersNM1001842 and M73238 (human); AF068615 (mouse); and S54212 (rat); eachof which is incorporated herein by reference.

TABLE 4 Alignment of human and mouse Cytokine-Like Factor 1 (CLF-1; SEQID NO: 12 and 13) See Elson, et al. (1998) J. Immunol. 161:1371-1379;GenBank Accession number AF059293 and NM_004750; also described byDouglas J. Hilton (Australia) in WO9920755. Reported signal sequence of37 amino acids in human form, cleavage at GSG/AHT. hCLF-1MPAGRRGPAAQSARRPPPLLPLLLLLCVLGAPRAGSGAHTAVISPQDPTL mCLF-1--------------RPLSSLWSPLLLCVLGVPRGGSGAHTAVISPQDPTL               ** .*   *******.**.**************** hCLF-1LIGSSLLATCSVHGDPPGATAEGLYWTLNGRRLPPELSRVLNASTLALAL mCLF-1LIGSSLQATCSIHGDTPGATAEGLYWTLNGRRLP-SLSRLLNTSTLALAL **********:***.****************** .***:**:******* hCLF-1ANLNGSRQRSGDNLVCHARDGSILAGSCLYVGLPPEKPVNISCWSKNMKD mCLF-1ANLNGSRQQSGDNLVCHARDGSILAGSCLYVGLPPEKPFNISCWSRNMKD********:*****************************.******.**** hCLF-1LTCRWTPGAHGETFLHTNYSLKYKLRWYGQDNTCEEYHTVGPHSCHIPKD mCLF-1LTCRWTPGAHGETFLHTNYSLKYKLRWYGQDNTCEEYHTVGPHSCHIPKD************************************************** hCLF-1LALFTPYEIWVEATNRLGSARSDVLTLDILDVVTTDPPPDVHVSRVGGLE mCLF-1LALFTPYEIWVEATNRLGSARSDVLTLDVLDVVTTDPPPDVHVSRVGGLE****************************:********************* hCLF-1DQLSVRWVSPPALKDFLFQAKYQIRYRVEDSVDWKVVDDVSNQTSCRLAG mCLF-1DQLSVRWVSPPALKDFLFQAKYQIRYRVEDSVDWKVVDDVSNQTSCRLAG************************************************** hCLF-1LKPGTVYFVQVRCNPFGIYGSKKAGIWSEWSHPTAASTPRSERPGPGGGA mCLF-1LKPGTVYFVQVRCNPFGIYGSKKAGIWSEWSHPTAASTPRSERPGPGGGV*************************************************. hCLF-1CEPRGGEPSSGPVRRELKQFLGWLKKHAYCSNLSFRLYDQWRAWMQKSHK mCLF-1CEPRGGEPSSGPVRRELKQFLGWLKKHAYCSNLSFRLYDQWRAWMQKSHK************************************************** hCLF-1TRNQ---VLPDKL--------- mCLF-1 TRNQDEGILPSGRRGAARGPAG ****   :**.

Standard domains of the human CLF-1 receptor sequence correspondapproximately to: signal from 1 to about 38; first IG-like domain fromabout residue 39 to 130; a second domain from about 131. to about 237;and the last from about 238 to the end.

The descriptions below may also be applied to the CLF-1, or to theIL-B60/CLF-1 complex. A fusion of the IL-B60 with CLF-1 may beconstructed, as, e.g., the hyper IL-6. See, e.g., Fischer, et al. (1997)Nature Biotechnol. 15:142-145; Rakemann, et al. (1999) J. Biol. Chem.274:1257-1266; and Peters, et al.(1998) J. Immunol. 161:3575-3581; whichare incorporated herein by reference.

The original discovery and molecular characterization of CNTF as apotent survival factor for neuronal cells (see, e.g., Hughes, et al.(1988) Nature 335:70-73; and Stockli, et al. (1989) Nature 342:920-923)suggested a prospective therapeutic use as a molecule that could speedrepair of damaged or severed motor neurons (Sendtner, et al. (1990)Nature 345:440-441; and Curtis, et al. (1993) Nature 365:253-256) orprevent nerve degeneration (Sendtner, et al. (1992) Nature 358:502-504;Emerich, et al. (1997) Nature 386:395-399; and Gravel, et al. (1997)Nature Med. 3:765-770). However, CNTF is oddly a protein without asecretory signal peptide, and does not appear to escape the cell(Stockli, et al., ibid); furthermore, engineered (Masu, et al. (1993)Nature 365:27-32) or naturally occurring (Takahashi, et al. (1994)Nature Genet. 7:79-84) disruptions of the CNTF gene are not deleterious.By contrast, gene disruptions of the primary receptor for CNTF (CNTF-Rα)prove lethal shortly after birth. DeChiara, et al. (1995) Cell83:313-322). Together, these observations point to the existence of asecond ligand for CNTF-Rα that is physiologically responsible for the invitro observed, or in vivo desired, actions of CNTF. This workdemonstrates that the composite cytokine IL-B60/CLF-1 is likely thislong sought-after factor that is both developmentally critical, it issecreted from target organs and directs their innervation by motorneurons, as well as therapeutically promising, since nerve transectionresults in a fast and. long lasting induction of both IL-B60 and CLF-1,indicating a role for the complex in regeneration. In support of thismodel, gene disruption of CLF-1 (Alexander, et al. (1999) Curr. Biol.9:605-608) is quite similar in phenotype to the CNTF-Rα knock-out.

In an intriguing twist, while IL-B60 has a signal peptide, its secretionremains critically dependent on complexing with CLF-1, as described.Once secreted, IL-B60 signals via a tripartite receptor system that isotherwise identical to that of CNTF, consisting of the two ubiquitouslyexpressed signal-transducing components gp130 and LIF-R, and thespecificity-determining receptor, CNTF-Rα. Strikingly, the role of CLF-1seems to be restricted to that of a chaperone, since it is discardedfrom the signaling complex after delivering IL-B60 to CNTF-Rα; indeed,the requirement for CLF-1 can be sidestepped by fusing IL-B60 directlyto a soluble form of the CNTF-Rα chain. All three protein chainsinvolved in this novel system, IL-B60, CLF-1, and CNTF-Rα, represent themost highly conserved sequences in the hematopoietic cytokine/receptorsuperfamily, indicating an evolutionarily critical interaction.Furthermore, the conditional use of a hematopoietic receptor as asecretion factor and escort presents a novel paradigm for cytokineactivity.

To summarize, this work sheds light on the entwined biological functionof two orphan molecules, IL-B60 and CLF-1, by describing their novelengagement of the CNTF receptor complex. In doing so, we present astrong argument that it is IL-B60/CLF-1 cytokine that serves as a keyphysiological factor in motor neuron development and regeneration.

II. Purified IL-B60 or Complex

Human IL-B60 amino acid sequence, is shown, in one embodiment within SEQID NO: 2. Other naturally occurring nucleic acids which encode theprotein can be isolated by standard procedures using the providedsequence, e.g., PCR techniques, or by hybridization. These amino acidsequences, provided amino to carboxy, are important in providingsequence information for the cytokine allowing for distinguishing theprotein antigen from other proteins and exemplifying numerous variants.Moreover, the peptide sequences allow preparation of peptides togenerate antibodies to recognize such segments, and nucleotide sequencesallow preparation of oligonucleotide probes, both of which arestrategies for detection or isolation, e.g., cloning, of genes encodingsuch sequences.

As used herein, the term “human soluble IL-B60” shall encompass, whenused in a protein context, a protein having amino acid sequencecorresponding to a soluble polypeptide from SEQ ID NO: 2. Significantfragments thereof will often retain similar functions, e.g.,antigenicity. Preferred embodiments comprise a plurality of distinct,e.g., nonoverlapping, segments of the specified length. Typically, theplurality will be at least two, more usually at least three, andpreferably 5, 7, or even more. While the length minima may be recited,longer lengths, of various sizes, may be appropriate, e.g., one oflength 7, and two of length 12. Similar features apply topolynucleotides.

Binding components, e.g., antibodies, typically bind to an IL-B60 withhigh affinity, e.g., at least about 100 nM, usually better than about 30nM, preferably better than about 10 nM, and more preferably at betterthan about 3 nM. Counterpart proteins will be found in mammalian speciesother than human, e.g., other primates, ungulates, or rodents.Non-mammalian species should also possess structurally or functionallyrelated genes and proteins, e.g., birds or amphibians.

The term “polypeptide” as used herein includes a significant fragment orsegment, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least about 12 amino acids, typicallyat least about 16 amino acids, preferably at least about 20 amino acids,and, in particularly preferred embodiments, at least about 30 or moreamino acids, e.g., 35, 40, 45, 50, etc. Such fragments may have endswhich begin and/or end at virtually all positions, e.g., beginning atresidues 1, 2, 3, etc., and ending at, e.g., 150, 149, 148, etc., in allpractical combinations. Particularly interesting peptides have endscorresponding to structural domain boundaries, e.g., helices A, B, C,and/or D. See Table 1.

The term “binding composition” refers to molecules that bind withspecificity to IL-B60, e.g., in an antibody-antigen interaction. Thespecificity may be more or less inclusive, e.g., specific to aparticular embodiment, or to groups of related embodiments, e.g.,primate, rodent, etc. Depletion or absorptions can provide desiredselectivities. Also provided are compounds, e.g., proteins, whichspecifically associate with IL-B60, including in a naturalphysiologically relevant protein-protein interaction, either covalent ornon-covalent. The molecule may be a polymer, or chemical reagent. Afunctional analog may be a protein with structural modifications, or itmay be a molecule which has a molecular shape which interacts with theappropriate binding determinants. The compounds may serve as agonists orantagonists of a receptor binding interaction, see, e.g., Goodman, etal. (eds.) Goodman & Gilman's: The Pharmacological Bases of Therapeutics(current ed.) Pergamon Press.

Substantially pure, e.g., in a protein context, typically means that theprotein is free from other contaminating proteins, nucleic acids, orother biologicals derived from the original source organism. Purity maybe assayed by standard methods, typically by weight, and will ordinarilybe at least about 40% pure, generally at least about 50% pure, often atleast about 60% pure, typically at least about 80% pure, preferably atleast about 90% pure, and in most preferred embodiments, at least about95% pure. Carriers or excipients will often be added.

Solubility of a polypeptide or fragment depends upon the environment andthe polypeptide. Many parameters affect polypeptide solubility,including temperature, electrolyte environment, size and molecularcharacteristics of the polypeptide, and nature of the solvent.Typically, the temperature at which the polypeptide is used ranges fromabout 4° C. to about 65° C. Usually the temperature at use is greaterthan about 18° C. For diagnostic purposes, the temperature will usuallybe about room temperature or warmer, but less than the denaturationtemperature of components in the assay. For therapeutic purposes, thetemperature will usually be body temperature, typically about 37° C. forhumans and mice, though under certain situations the temperature may beraised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be in asubstantially stable state, and usually not in a denatured state. Thepolypeptide may be associated with other polypeptides in a quaternarystructure, e.g., to confer solubility, or associated with lipids ordetergents.

The solvent and electrolytes will usually be a biologically compatiblebuffer, of a type used for preservation of biological activities, andwill usually approximate a physiological aqueous solvent. Usually thesolvent will have a neutral pH, typically between about 5 and 10, andpreferably about 7.5. On some occasions, one or more detergents will beadded, typically a mild non-denaturing one, e.g., CHS (cholesterylhemisuccinate) or CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), or a low enough concentration as to avoid significantdisruption of structural or physiological properties of the protein. Inother instances, a harsh detergent may be used to effect significantdenaturation.

An IL-B60 polypeptide that specifically binds to or that is specificallyimmunoreactive with an antibody, e.g., such as a polyclonal antibody,generated against a defined immunogen, e.g., such as an immunogenconsisting of an amino acid sequence of SEQ ID NO: 2 or fragmentsthereof or a polypeptide generated from the nucleic acid of SEQ ID NO: 1is typically determined in an immunoassay. Included within the metes andbounds of the present invention are those nucleic acid sequencesdescribed herein, including functional variants, that encodepolypeptides that selectively bind to polyclonal antibodies generatedagainst the prototypical IL-B60 polypeptide as structurally andfunctionally defined herein. The immunoassay typically uses a polyclonalantiserum which was raised, e.g., to a protein of SEQ ID NO: 2. Thisantiserum is selected, or depleted, to have low crossreactivity againstappropriate other closely related family members, preferably from thesame species, and any such crossreactivity is removed byimmunoabsorption or depletion prior to use in the immunoassay.Appropriate selective serum preparations can be isolated, andcharacterized.

In order to produce antisera for use in an immunoassay, the protein,e.g., of SEQ ID NO: 2, is isolated as described herein. For example,recombinant protein may be produced in a mammalian cell line. Anappropriate host, e.g., an inbred strain of mice such as Balb/c, isimmunized with the protein of SEQ ID NO: 2 using a standard adjuvant,such as Freund's adjuvant, and a standard mouse immunization protocol(see Harlow and Lane). Alternatively, a substantially full lengthsynthetic peptide derived from the sequences disclosed herein can beused as an immunogen. Polyclonal sera are collected and titered againstthe immunogen protein in an immunoassay, e.g., a solid phase immunoassaywith the immunogen immobilized on a solid support. Polyclonal antiserawith a titer of 10⁴ or greater are selected and tested for their crossreactivity against other closely related family members, e.g., LIF,CT-1, CNTF, DIL-40, or other members of the IL-6 family, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573. Preferably at least two IL-6/IL-12 familymembers are used in this determination in conjunction with the target.These long chain cytokine family members can be produced as recombinantproteins and isolated using standard molecular biology and proteinchemistry techniques as described herein. Thus, antibody preparationscan be identified or produced having desired selectivity or specificityfor subsets of IL-B60 family members. Alternatively, antibodies may beprepared which bind to the complex comprising the IL-B60 with the CLF-1.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the protein of SEQ ID NO: 2can be immobilized to a solid support. Proteins added to the assaycompete with the binding of the antisera to the immobilized antigen. Theability of the above proteins to compete with the binding of theantisera to the immobilized protein is compared to the protein of SEQ IDNO: 2. The percent crossreactivity for the above proteins is calculated,using standard calculations. Those antisera with less than 10%crossreactivity with each of the proteins listed above are selected andpooled. The cross-reacting antibodies are then removed from the pooledantisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein. In order to make this comparison, the twoproteins are each assayed at a wide range of concentrations and theamount of each protein required to inhibit 50% of the binding of theantisera to the immobilized protein is determined. If the amount of thesecond protein required is less than twice the amount of the protein of,e.g., SEQ ID NO: 2 that is required, then the second protein is said tospecifically bind to an antibody generated to the immunogen.

III. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence identity with the amino acid sequence of the IL-B60antigen. The variants include species, polymorphic, or allelic variants.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Chapter One in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group, Madison, Wis. Sequence identity changes whenconsidering conservative substitutions as matches. Conservativesubstitutions typically include substitutions within the followinggroups: glycine, alanine; valine, isoleucine, leucine; aspartic acid,glutamic acid; asparagine, glutamine; serine, threonine; lysine,arginine; and phenylalanine, tyrosine. The conservation may apply tobiological features, functional features, or structural features.Homologous amino acid sequences are typically intended to includenatural polymorphic or allelic and interspecies variations of a proteinsequence. Typical homologous proteins or peptides will have from 25-100%identity (if gaps can be introduced), to 50-100% identity (ifconservative substitutions are included) with the amino acid sequence ofthe IL-B60. Identity measures will be at least about 35%, generally atleast about 40%, often at least about 50%, typically at least about 60%,usually at least about 70%, preferably at least about 80%, and morepreferably at least about 90%.

The isolated IL-B60 DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, andinversions of short nucleotide stretches. These modifications result innovel DNA sequences which encode these antigens, their derivatives, orproteins having similar physiological, immunogenic, antigenic, or otherfunctional activity. These modified sequences can be used to producemutant antigens or to enhance expression. Enhanced expression mayinvolve gene amplification, increased transcription, increasedtranslation, and other mechanisms. “Mutant IL-B60” encompasses apolypeptide otherwise falling within the sequence identity definition ofthe IL-B60 as set forth above, but having an amino acid sequence whichdiffers from that of IL-B60 as normally found in nature, whether by wayof deletion, substitution, or insertion. This generally includesproteins having significant identity with a protein having sequence ofSEQ ID NO: 2, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most of the natural full length disclosed sequences.Full length sequences will typically be preferred, though truncatedversions will also be useful, likewise, genes or proteins found fromnatural sources are typically most desired. Similar concepts apply todifferent IL-B60 proteins, particularly those found in various warmblooded animals, e.g., mammals and birds. These descriptions aregenerally meant to encompass all IL-B60 proteins, not limited to theparticular primate embodiments specifically discussed.

IL-B60 mutagenesis can also be conducted by making amino acid insertionsor deletions. Substitutions, deletions, insertions, or any combinationsmay be generated to arrive at a final construct. Insertions includeamino- or carboxy-terminal fusions. Random mutagenesis can be conductedat a target codon and the expressed mutants can then be screened for thedesired activity. Methods for making substitution mutations atpredetermined sites in DNA having a known sequence are well known in theart, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR)techniques. See, e.g., Sambrook, et al. (1989); Ausubel, et al. (1987and Supplements); and Kunkel, et al. (1987) Methods in Enzymol.154:367-382. Preferred embodiments include, e.g., 1-fold, 2-fold,3-fold, 5-fold, 7-fold, etc., preferably conservative substitutions atthe nucleotide or amino acid levels. Preferably the substitutions willbe away from the conserved cysteines, and often will be in the regionsaway from the helical structural domains. Such variants may be useful toproduce specific antibodies, and often will share many or all biologicalproperties. See Table 2. Recognition of the cytokine structure providesimportant insight into the structure and positions of residues which maybe modified to effect desired changes in receptor interaction. Also, theinteraction of the IL-B60 with the CLF-1 protein requires complementarystructural features in the interacting surface.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins. Aheterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. A similar conceptapplies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, target-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence, e.g., PCRtechniques.

Structural analysis can be applied to this gene, in comparison to theIL-6 family of cytokines. The family includes, e.g., IL-6, IL-11, IL-12,G-CSF, LIF, OSM, CNTF, and Ob. Alignment of the human and mouse IL-B60sequences with other members of the IL-6 family should allow definitionof structural features. In particular, β-sheet and α-helix residues canbe determined using, e.g., RASMOL program, see Bazan, et al. (1996)Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle andMilner-White (1995) TIBS 20:374-376; and Gronenberg, et al. (1991)Protein Engineering 4:263-269. See, also, Wilkins, et al. (eds. 1997)Proteome Research: New Frontiers in Functional Genomics Springer-Verlag,NY. Preferred residues for substitutions include the surface exposedresidues which would be predicted to interact with receptor. Otherresidues which should conserve function will be conservativesubstitutions, particularly at position far from the surface exposedresidues.

IV. Functional Variants

The blocking of physiological response to IL-B60s may result from thecompetitive inhibition of binding of the ligand to its receptor.

In vitro assays of the present invention will often use isolatedprotein, soluble fragments comprising receptor binding segments of theseproteins, or fragments attached to solid phase substrates. These assayswill also allow for the diagnostic determination of the effects ofeither binding segment mutations and modifications, or cytokinemutations and modifications, e.g., IL-B60 analogs.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the cytokine, or receptorbinding fragments compete with a test compound.

“Derivatives” of IL-B60 antigens include amino acid sequence mutantsfrom naturally occurring forms, glycosylation variants, and covalent oraggregate conjugates with other chemical moieties. Covalent derivativescan be prepared by linkage of functionalities to groups which are foundin IL-B60 amino acid side chains or at the N- or C-termini, e.g., bystandard means. See, e.g., Lundblad and Noyes (1988) Chemical Reagentsfor Protein Modification, vols. 1-2, CRC Press, Inc., Boca Raton, Fla.;Hugli (ed. 1989) Techniques in Protein Chemistry, Academic Press, SanDiego, Calif.; and Wong (1991) Chemistry of Protein Conjugation andCross Linking, CRC Press, Boca Raton, Fla.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. See, e.g.,Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versionsof the peptides with the same primary amino acid sequence which haveother minor modifications, including phosphorylated amino acid residues,e.g., phosphotyrosine, phosphoserine, or phosphothreonine.

Fusion polypeptides between IL-B60s and other homologous or heterologousproteins are also provided. Many cytokine receptors or other surfaceproteins are multimeric, e.g., homodimeric entities, and a repeatconstruct may have various advantages, including lessened susceptibilityto proteolytic cleavage. Typical examples are fusions of a reporterpolypeptide, e.g., luciferase, with a segment or domain of a protein,e.g., a receptor-binding segment, so that the presence or location ofthe fused ligand may be easily determined. See, e.g., Dull, et al., U.S.Pat. No. 4,859,609. Other gene fusion partners include bacterialβ-galactosidase, trpE, Protein A, β-lactamase, alpha amylase, alcoholdehydrogenase, yeast alpha mating factor, and detection or purificationtags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, etal. (1988) Science 241:812-816.

Fusion peptides will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.),vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al. (eds.1993) Current Protocols in Molecular Biology, Greene and Wiley, NY.Techniques for synthesis of polypeptides are described, e.g., inMerrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; and Grant (1992)Synthetic Peptides: A User's Guide, W. H. Freeman, NY. Refolding methodsmay be applicable to synthetic proteins.

This invention also contemplates the use of derivatives of IL-B60proteins other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties or protein carriers. Covalent or aggregativederivatives will be useful as immunogens, as reagents in immunoassays,or in purification methods such as for affinity purification of bindingpartners, e.g., other antigens. An IL-B60 can be immobilized by covalentbonding to a solid support such as cyanogen bromide-activated SEPHAROSE,by methods which are well known in the art, or adsorbed onto polyolefinsurfaces, with or without glutaraldehyde cross-linking, for use in theassay or purification of anti-IL-B60 antibodies or an alternativebinding composition. The IL-B60 proteins can also be labeled with adetectable group, e.g., for use in diagnostic assays. Purification ofIL-B60 may be effected by an immobilized antibody or complementarybinding partner, e.g., binding portion of a receptor.

A solubilized IL-B60 or fragment of this invention can be used as animmunogen for the production of antisera or antibodies specific forbinding. Purified antigen can be used to screen monoclonal antibodies orantigen-binding fragments, encompassing antigen binding fragments ofnatural antibodies, e.g., Fab, Fab′, F(ab)2, etc. Purified IL-B60antigens can also be used as a reagent to detect antibodies generated inresponse to the presence of elevated levels of the cytokine, which maybe diagnostic of an abnormal or specific physiological or diseasecondition. This invention contemplates antibodies raised against aminoacid sequences encoded by nucleotide sequence shown in SEQ ID NO: 1, orfragments of proteins containing it. In particular, this inventioncontemplates antibodies having binding affinity to or being raisedagainst specific domains, e.g., helices A, B, C, or D of the IL-B60, orthe Ig domains of the CLF-1.

The present invention contemplates the isolation of additional closelyrelated species variants. Southern and Northern blot analysis willestablish that similar genetic entities exist in other mammals. It islikely that IL-B60s are widespread in species variants, e.g., rodents,lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.

The invention also provides means to isolate a group of related antigensdisplaying both distinctness and similarities in structure, expression,and function. Elucidation of many of the physiological effects of themolecules will be greatly accelerated by the isolation andcharacterization of additional distinct species or polymorphic variantsof them. In particular, the present invention provides useful probes foridentifying additional homologous genetic entities in different species.

The isolated genes will allow transformation of cells lacking expressionof an IL-B60, e.g., either species types or cells whichlack-corresponding proteins and exhibit negative background activity.This should allow analysis of the function of IL-B60 in comparison tountransformed control cells.

Dissection of critical structural elements which effect the variousphysiological functions mediated through these antigens is possibleusing standard techniques of modern molecular biology, particularly incomparing members of the related class. See, e.g., the homolog-scanningmutagenesis technique described in Cunningham, et al. (1989) Science243:1339-1336; and approaches used in O'Dowd, et al. (1988) J. Biol.Chem. 263:15985-15992; and Lechleiter, et al. (1990) EMBO J.9:4381-4390.

Intracellular functions would probably involve receptor signaling.However, protein internalization may occur under certain circumstances,and interaction between intracellular components and cytokine may occur.Specific segments of interaction of IL-B60 with interacting componentsmay be identified by mutagenesis or direct biochemical means, e.g.,cross-linking or affinity methods. Structural analysis bycrystallographic or other physical methods will also be applicable.Further investigation of the mechanism of signal transduction willinclude study of associated components which may be isolatable byaffinity methods or by genetic means, e.g., complementation analysis ofmutants.

Further study of the expression and control of IL-B60 will be pursued.The controlling elements associated with the antigens should exhibitdifferential physiological, developmental, tissue specific, or otherexpression patterns. Upstream or downstream genetic regions, e.g.,control elements, are of interest.

Structural studies of the IL-B60 antigens will lead to design of newantigens, particularly analogs exhibiting agonist or antagonistproperties on the molecule. This can be combined with previouslydescribed screening methods to isolate antigens exhibiting desiredspectra of activities.

V. Antibodies

Antibodies can be raised to various epitopes of the IL-B60 proteins,including species, polymorphic, or allelic variants, and fragmentsthereof, both in their naturally occurring forms and in theirrecombinant forms. Additionally, antibodies can be raised to IL-B60s ineither their active forms or in their inactive forms, including nativeor denatured versions. Anti-idiotypic antibodies are also contemplated.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the antigens can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective IL-B60s, or screened for agonistic orantagonistic activity, e.g., mediated through a receptor. Antibodies maybe agonistic or antagonistic, e.g., by sterically blocking binding to areceptor. These monoclonal antibodies will usually bind with at least aK_(D) of about 1 mM, more usually at least about 300 μM, typically atleast about 100 μM, more typically at least about 30 μM, preferably atleast about 10 μM, and more preferably at least about 3 μM or better.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bescreened for ability to bind to the antigens without inhibiting bindingto a receptor. As neutralizing antibodies, they can be useful incompetitive binding assays. They will also be useful in detecting orquantifying IL-B60 protein or its receptors. See, e.g., Chan (ed. 1987)Immunology: A Practical Guide, Academic Press, Orlando, Fla.; Price andNewman (eds. 1991) Principles and Practice of Immunoassay, StocktonPress, N.Y.; and Ngo (ed. 1988) Nonisotopic Immunoassay, Plenum Press,N.Y. Cross absorptions or other tests will identify antibodies whichexhibit various spectra of specificities, e.g., unique or shared speciesspecificities.

Further, the antibodies, including antigen binding fragments, of thisinvention can be potent antagonists that bind to the antigen and inhibitfunctional binding, e.g., to a receptor which may elicit a biologicalresponse. They also can be useful as non-neutralizing antibodies and canbe coupled to toxins or radionuclides so that when the antibody binds toantigen, a cell expressing it, e.g., on its surface, is killed. Further,these antibodies can be conjugated to drugs or other therapeutic agents,either directly or indirectly by means of a linker, and may effect drugtargeting.

Antigen fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. An antigen and its fragments may be fused or covalentlylinked to a variety of immunogens, such as keyhole limpet hemocyanin,bovine serum albumin, tetanus toxoid, etc. See Microbiology, HoeberMedical Division, Harper and Row, 1969; Landsteiner (1962) Specificityof Serological Reactions, Dover Publications, New York; Williams, et al.(1967) Methods in Immunology and Immunochemistry, vol. 1, AcademicPress, New York; and Harlow and Lane (1988) Antibodies: A LaboratoryManual, CSH Press, NY, for descriptions of methods of preparingpolyclonal antisera.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.), Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546. The polypeptides and antibodies of thepresent invention may be used with or without modification, includingchimeric or humanized antibodies. Frequently, the polypeptides andantibodies will be labeled by joining, either covalently ornon-covalently, a substance which provides for a detectable signal. Awide variety of labels and conjugation techniques are known and arereported extensively in both the scientific and patent literature.Suitable labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent moieties, chemiluminescent moieties, magneticparticles, and the like. Patents, teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulinsmay be produced, see Cabilly, U.S. Pat. No. 4,816,567; Moore, et al.,U.S. Pat. No. 4,642,334; and Queen, et al. (1989) Proc. Nat'l Acad. Sci.USA 86:10029-10033.

The antibodies of this invention can also be used for affinitychromatography in isolating the protein. Columns can be prepared wherethe antibodies are linked to a solid support. See, e.g., Wilchek et al.(1984) Meth. Enzymol. 104:3-55. Conversely, protein can be used fordepletion or cross absorptions to prepare selectively specific bindingcompositions.

Antibodies raised against each IL-B60 will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

VI. Nucleic Acids

The described peptide sequences and the related reagents are useful indetecting, isolating, or identifying a DNA clone encoding IL-B60, e.g.,from a natural source. Typically, it will be useful in isolating a genefrom a mammal, and similar procedures will be applied to isolate genesfrom other species, e.g., warm blooded animals, such as birds andmammals. Cross hybridization will allow isolation of IL-B60 from thesame, e.g., polymorphic variants, or other species. A number ofdifferent approaches will be available to successfully isolate asuitable nucleic acid clone.

The purified protein or polypeptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein can be presented to an immune system to generatemonoclonal or polyclonal antibodies. See, e.g., Coligan (1991) CurrentProtocols in Immunology Wiley/Greene; and Harlow and Lane (1989)Antibodies: A Laboratory Manual, Cold Spring Harbor Press.

For example, a specific binding composition could be used for screeningof an expression library made from a cell line which expresses anIL-B60. Screening of intracellular expression can be performed byvarious staining or immunofluorescence procedures. Binding compositionscould be used to affinity purify or sort out cells expressing a surfacefusion protein.

The peptide segments can also be used to predict appropriateoligonucleotides to screen a library. The genetic code can be used toselect appropriate oligonucleotides useful as probes for screening. See,e.g., SEQ ID NO: 1. In combination with polymerase chain reaction (PCR)techniques, synthetic oligonucleotides will be useful in selectingcorrect clones from a library. Complementary sequences will also be usedas probes, primers, or antisense strands. Various fragments should beparticularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

This invention contemplates use of isolated DNA or fragments to encode abiologically active corresponding IL-B60 polypeptide, particularlylacking the portion coding the untranslated portions of the describedsequence. In addition, this invention covers isolated or recombinant DNAwhich encodes a biologically active protein or polypeptide and which iscapable of hybridizing under appropriate conditions with the DNAsequences described herein. Said biologically active protein orpolypeptide can be an intact antigen, or fragment, and have an aminoacid sequence disclosed in, e.g., SEQ ID NO: 2, particularly a mature,secreted-polypeptide. Further, this invention covers the use of isolatedor recombinant DNA, or fragments thereof, which encode proteins whichexhibit high identity to a secreted IL-B60. The isolated DNA can havethe respective regulatory sequences in the 5′ and 3′ flanks, e.g.,promoters, enhancers, poly-A addition signals, and others.Alternatively, expression may be effected by operably linking a codingsegment to a heterologous promoter, e.g., by inserting a promoterupstream from an endogenous gene.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other componentswhich naturally accompany a native sequence, e.g., ribosomes,polymerases, and/or flanking genomic sequences from the originatingspecies. The term embraces a nucleic acid sequence which has beenremoved from its naturally occurring environment, and includesrecombinant or cloned DNA isolates and chemically synthesized analogs oranalogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule.Generally, the nucleic acid will be in a vector or fragment less thanabout 50 kb, usually less than about 30 kb, typically less than about 10kb, and preferably less than about 6 kb.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain minor heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, e.g., products made by transforming cells with anyunnaturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site.

Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a single genetic entity comprising adesired combination of functions not found in the commonly availablenatural forms. Restriction enzyme recognition sites are often the targetof such artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. Specifically included are synthetic nucleic acids which, bygenetic code redundancy, encode polypeptides similar to fragments ofthese antigens, and fusions of sequences from various different speciesor polymorphic variants.

A significant “fragment” in a nucleic acid context is a contiguoussegment of at least about 17 nucleotides, generally at least about 22nucleotides, ordinarily at least about 29 nucleotides, more often atleast about 35 nucleotides, typically at least about 41 nucleotides,usually at least about 47 nucleotides, preferably at least about 55nucleotides, and in particularly preferred embodiments will be at leastabout 60 or more nucleotides, e.g., 67, 73, 81, 89, 95, etc.

A DNA which codes for an IL-B60 protein will be particularly useful toidentify genes, mRNA, and cDNA species which code for related or similarproteins; as well as DNAs which code for homologous proteins fromdifferent species. There will be homologs in other species, includingprimates, rodents, canines, felines, and birds. Various IL-B60 proteinsshould be homologous and are encompassed herein. However, even proteinsthat have a more distant evolutionary relationship to the antigen canreadily be isolated under appropriate conditions using these sequencesif they are sufficiently homologous. Primate IL-B60 proteins are ofparticular interest.

Recombinant clones derived from the genomic sequences, e.g., containingintrons, will be useful for transgenic studies, including, e.g.,transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow(1992) “Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology,Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989)Science 244:1288; Robertson (ed. 1987) Teratocarcinomas and EmbryonicStem Cells: A Practical Approach, IRL Press, Oxford; and Rosenberg(1992) J. Clinical Oncology 10:180-199.

Substantial homology, e.g., identity, in the nucleic acid sequencecomparison context means either that the segments, or theircomplementary strands, when compared, are identical when optimallyaligned, with appropriate nucleotide insertions or deletions, in atleast about 50% of the nucleotides, generally at least about 58%,ordinarily at least about 65%, often at least about 71%, typically atleast about 77%, usually at least about 85%, preferably at least about95 to 98% or more, and in particular embodiments, as high as about 99%or more of the nucleotides. Alternatively, substantial homology existswhen the segments will hybridize under selective hybridizationconditions, to a strand, or its complement, typically using a sequenceof IL-B60, e.g., in SEQ ID NO: 1. Typically, selective hybridizationwill occur when there is at least about 55% identity over a stretch ofat least about 30 nucleotides, preferably at least about 75% over astretch of about 25 nucleotides, and most preferably at least about 90%over about 20 nucleotides. See, Kanehisa (1984) Nuc. Acids Res.12:203-213. The length of identity comparison, as described, may be overlonger stretches, and in certain embodiments will be over a stretch ofat least about 17 nucleotides, usually at least about 28 nucleotides,typically at least about 40 nucleotides, and preferably at least about75 to 100 or more nucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters, typically those controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30° C., usually in excess ofabout 37° C., typically in excess of about 55° C., preferably in excessof about 70° C. Stringent salt conditions will ordinarily be less thanabout 1000 mM, usually less than about 400 mM, typically less than about250 mM, preferably less than about 150 mM, including about 100, 50, oreven 20 mM. However, the combination of parameters is much moreimportant than the measure of any single parameter. See, e.g., Wetmurand Davidson (1968) J. Mol. Biol. 31:349-370. Hybridization understringent conditions should give background of at least 2-fold overbackground, preferably at least 3-5 or more.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optical alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith and Waterman (1981) Adv. Appl.Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol. 48:443, by the search for similarity method ofPearson and Lipman (1988) Proc. Nat'l Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by visual inspection (seegenerally Ausubel et al., supra).

One example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments to show relationship and percent sequence identity.It also plots a tree or dendrogram showing the clustering relationshipsused to create the alignment. PILEUP uses a simplification of theprogressive alignment method of Feng and Doolittle (1987) J. Mol. Evol.35:351-360. The method used is similar to the method described byHiggins and Sharp (1989) CABIOS 5:151-153. The program can align up to300 sequences, each of a maximum length of 5,000 nucleotides or aminoacids. The multiple alignment procedure begins with the pairwisealignment of the two most similar sequences, producing a cluster of twoaligned sequences. This cluster is then aligned to the next most relatedsequence or cluster of aligned sequences. Two clusters of sequences arealigned by a simple extension of the pairwise alignment of twoindividual sequences. The final alignment is achieved by a series ofprogressive, pairwise alignments. The program is run by designatingspecific sequences and their amino acid or nucleotide coordinates forregions of sequence comparison and by designating the programparameters. For example, a reference sequence can be compared to othertest sequences to determine the percent sequence identity relationshipusing the following parameters: default gap weight (3.00), default gaplength weight (0.10), and weighted end gaps.

Another example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described Altschul, et al. (1990) J. Mol. Biol. 215:403-410. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http:www.ncbi.nlm.nih.gov/). Thisalgorithm involves first identifying high scoring sequence pairs (HSPs)by identifying short words of length W in the query sequence, whicheither match or satisfy some positive-valued threshold score T whenaligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul, et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Extension of the word hitsin each direction are halted when: the cumulative alignment score fallsoff by the quantity X from its maximum achieved value; the cumulativescore goes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLAST program uses asdefaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (seeHenikoff and Henikoff (1989) Proc. Nat'l Acad. Sci. USA 89:10915)alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparisonof both strands.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad.Sci. USA 90:5873-5787). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences of polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

IL-B60 from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. Homology may berelatively low between distantly related species, and thus hybridizationof relatively closely related species is advisable. Alternatively,preparation of an antibody preparation which exhibits less speciesspecificity may be useful in expression cloning approaches.

VII. Making IL-B60 or Complex; Mimetics

DNA which encodes the IL-B60 or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gublerand Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNA Cloning: APractical Approach, IRL Press, Oxford. Alternatively, the sequencesprovided herein provide useful PCR primers or allow synthetic or otherpreparation of suitable genes encoding an IL-B60; including naturallyoccurring embodiments.

This DNA can be expressed in a wide variety of host cells for thesynthesis of a full-length IL-B60 or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual, Elsevier, N.Y.; and Rodriguez, et al. (eds. 1988) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses, Buttersworth,Boston, Mass.

For purposes of this invention, DNA sequences are operably linked whenthey are functionally related to each other. For example, DNA for apresequence or secretory leader is operably linked to a polypeptide ifit is expressed as a preprotein or participates in directing thepolypeptide to the cell membrane or in secretion of the polypeptide. Apromoter is operably linked to a coding sequence if it controls thetranscription of the polypeptide; a ribosome binding site is operablylinked to a coding sequence if it is positioned to permit translation.Usually, operably linked means contiguous and in reading frame, however,certain genetic elements such as repressor genes are not contiguouslylinked but still bind to operator sequences that in turn controlexpression. See, e.g., Rodriguez, et al., Chapter 10, pp. 205-236;Balbas and Bolivar (1990) Methods in Enzymology 185:14-37; and Ausubel,et al. (1993) Current Protocols in Molecular Biology, Greene and Wiley,NY.

Representative examples of suitable expression vectors include pCDNA1;pCD, see Okayama, et al. (1985) Mol. Cell Biol. 5:1136-1142; pMC1neoPoly-A, see Thomas, et al. (1987) Cell 51:503-512; and a baculovirusvector such as pAC 373 or pAC 610. See, e.g., Miller (1988) Ann. Rev.Microbiol. 42:177-199.

It will often be desired to express an IL-B60 polypeptide in a systemwhich provides a specific or defined glycosylation pattern. See, e.g.,Luckow and Summers (1988) Bio/Technology 6:47-55; and Kaufman (1990)Meth. Enzymol. 185:487-511.

The IL-B60, or a fragment thereof, may be engineered to be phosphatidylinositol (PI) linked to a cell membrane, but can be removed frommembranes by treatment with a phosphatidyl inositol cleaving enzyme,e.g., phosphatidyl inositol phospholipase-C. This releases the antigenin a biologically active form, and allows purification by standardprocedures of protein chemistry. See, e.g., Low (1989) Biochim. Biophys.Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,et al. (1991) J. Cell Biol. 114:1275-1283.

Now that the IL-B60 has been characterized, fragments or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis, Springer-Verlag, New York; Bodanszky (1984) The Principles ofPeptide Synthesis, Springer-Verlag, New York; and Villafranca (ed. 1991)Techniques in Protein Chemistry II, Academic Press, San Diego, Calif.

VIII. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in IL-B60mediated conditions, or below in the description of kits for diagnosis.The gene may be useful in forensic sciences, e.g., to distinguish rodentfrom human, or as a marker to distinguish between different cellsexhibiting differential expression or modification patterns. Theprovided compositions are useful reagents for, e.g., in vitro assays,scientific research, and the synthesis or manufacture of nucleic acids,polypeptides, or antibodies.

This invention also provides reagents with significant commercial and/ortherapeutic potential. The IL-B60 (naturally occurring or recombinant),fragments thereof, and antibodies thereto, along with compoundsidentified as having binding affinity to IL-B60, should be useful asreagents for teaching techniques of molecular biology, immunology, orphysiology. Appropriate kits may be prepared with the reagents, e.g., inpractical laboratory exercises in production or use of proteins,antibodies, cloning methods, histology, etc.

The reagents will also be useful in the treatment of conditionsassociated with abnormal physiology or development, includinginflammatory conditions. They may be useful in vitro tests for presenceor absence of interacting components, which may correlate with successof particular treatment strategies. In particular, modulation ofphysiology of various, e.g., hematopoietic or lymphoid, cells will beachieved by appropriate methods for treatment using the compositionsprovided herein. See, e.g., Thomson (1994; ed.) The Cytokine Handbook(2d ed.) Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

For example, a disease or disorder associated with abnormal expressionor abnormal signaling by an IL-B60 should be a likely target for anagonist or antagonist. The new cytokine should play a role in regulationor development of hematopoietic cells, e.g., lymphoid cells, whichaffect immunological responses, e.g., inflammation and/or autoimmunedisorders. Alternatively, it may affect vascular physiology ordevelopment, or neuronal effects.

In particular, the cytokine should mediate, in various contexts,cytokine synthesis by the cells, proliferation, etc. Antagonists ofIL-B60, such as mutein variants of a naturally occurring form of IL-B60or blocking antibodies, may provide a selective and powerful way toblock immune responses, e.g., in situations as inflammatory orautoimmune responses. See also Samter, et al. (eds.) ImmunologicalDiseases vols. 1 and 2, Little, Brown and Co.

In addition, certain combination compositions would be useful, e.g.,with other modulators of inflammation. Such other molecules may includesteroids, other versions of IL-6 and/or G-CSF, including speciesvariants, or viral homologs, and their respective antagonists.

Various abnormal conditions are known in each of the cell types shown toproduce IL-B60 mRNA by Northern blot analysis. See Berkow (ed.) TheMerck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; Thorn,et al. Harrison's Principles of Internal Medicine, McGraw-Hill, N.Y.;and Weatherall, et al. (eds.) Oxford Textbook of Medicine, OxfordUniversity Press, Oxford. Many other medical conditions and diseasesinvolve activation by macrophages or monocytes, and many of these willbe responsive to treatment by an agonist or antagonist provided herein.See, e.g., Stites and Terr (eds.; 1991) Basic and Clinical ImmunologyAppleton and Lange, Norwalk, Conn.; and Samter, et al. (eds.)Immunological Diseases Little, Brown and Co. These problems should besusceptible to prevention or treatment using compositions providedherein. The pancreatic islet localization suggests a possible relevanceto diabetes.

IL-B60, antagonists, antibodies, etc., can be purified and thenadministered to a patient, veterinary or human. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers, excipients, or preservatives. These combinationscan be sterile filtered and placed into dosage forms as bylyophilization in dosage vials or storage in stabilized aqueouspreparations. This invention also contemplates use of antibodies orbinding fragments thereof, including forms which are not complementbinding.

Drug screening using IL-B60 or fragments thereof can be performed toidentify compounds having binding affinity to or other relevantbiological effects on IL-B60 functions, including isolation ofassociated components. Subsequent biological assays can then be utilizedto determine if the compound has intrinsic stimulating activity and istherefore a blocker or antagonist in that it blocks the activity of thecytokine. Likewise, a compound having intrinsic stimulating activity canactivate the signal pathway and is thus an agonist in that it simulatesthe activity of IL-B60. This invention further contemplates thetherapeutic use of blocking antibodies to IL-B60 as antagonists and ofstimulatory antibodies as agonists. This approach should be particularlyuseful with other IL-B60 species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17thed. (1990), Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 pM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous or long term administration. See, e.g., Langer (1990) Science249:1527-1533.

IL-B60, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, topical, or parenteral (including subcutaneous, intramuscular,intravenous and intradermal) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethods well known in the art of pharmacy. See, e.g., Gilman, et al.(eds. 1990) Goodman and Gilman's: The Pharmacological Bases ofTherapeutics, 8th Ed., Pergamon Press; and Remington's PharmaceuticalSciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; Avis, etal. (eds. 1993) Pharmaceutical Dosage Forms: Parenteral Medications,Dekker, New York; Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Tablets, Dekker, New York; and Lieberman, et al. (eds. 1990)Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York. Thetherapy of this invention may be combined with or used in associationwith other agents, e.g., other cytokines, including IL-6 or G-CSF, ortheir respective antagonists.

Both naturally occurring and recombinant forms of the IL-B60s of thisinvention are particularly useful in kits and assay methods which arecapable of screening compounds for binding activity to the proteins.Several methods of automating assays have been developed in recent yearsso as to permit screening of tens of thousands of compounds in a shortperiod. See, e.g., Fodor, et al. (1991) Science 251:767-773, whichdescribes means for testing of binding affinity by a plurality ofdefined polymers synthesized on a solid substrate. The development ofsuitable assays can be greatly facilitated by the availability of largeamounts of purified, soluble IL-B60 as provided by this invention.

Other methods can be used to determine the critical residues inIL-B60-IL-B60 receptor interactions. Mutational analysis can beperformed, e.g., see Somoza, et al. (1993) J. Exptl. Med. 178:549-558,to determine specific residues critical in the interaction and/orsignaling. PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (Kingand Sternberg (1996) Protein Sci. 5:2298-2310) can provide secondarystructure predictions of α-helix (H), β-strand (E), or coil (L). HelicesA and D are most important in receptor interaction, with the D helix themore important region. See Table 2.

For example, antagonists can normally be found once the antigen has beenstructurally defined, e.g., by tertiary structure data. Testing ofpotential interacting analogs is now possible upon the development ofhighly automated assay methods using a purified IL-B60. In particular,new agonists and antagonists will be discovered by using screeningtechniques described herein. Of particular importance are compoundsfound to have a combined binding affinity for a spectrum of IL-B60molecules, e.g., compounds which can serve as antagonists for speciesvariants of IL-B60.

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing an IL-B60. Cells may be isolated which express an IL-B60 inisolation from other molecules. Such cells, either in viable or fixedform, can be used for standard binding partner binding assays. See also,Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990)Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto an IL-B60 and is described in detail in Geysen, European PatentApplication 84/03564, published on Sep. 13, 1984. First, large numbersof different small peptide test compounds are synthesized on a solidsubstrate, e.g., plastic pins or some other appropriate surface, seeFodor, et al. (1991). Then all the pins are reacted with solubilized,unpurified or solubilized, purified IL-B60, and washed. The next stepinvolves detecting bound IL-B60.

Rational drug design may also be based upon structural studies of themolecular shapes of the IL-B60 and other effectors or analogs. Effectorsmay be other proteins which mediate other functions in response tobinding, or other proteins which normally interact with IL-B60, e.g., areceptor. One means for determining which sites interact with specificother proteins is a physical structure determination, e.g., x-raycrystallography or 2 dimensional NMR techniques. These will provideguidance as to which amino acid residues form molecular contact regions,as modeled, e.g., against other cytokine-receptor models. For a detaileddescription of protein structural determination, see, e.g., Blundell andJohnson (1976) Protein Crystallography, Academic Press, New York.

IX. Kits

This invention also contemplates use of IL-B60 proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of another IL-B60 or bindingpartner. Typically the kit will have a compartment containing either adefined IL-B60 peptide or gene segment or a reagent which recognizes oneor the other, e.g., IL-B60 fragments or antibodies.

A kit for determining the binding affinity of a test compound to anIL-B60 would typically comprise a test compound; a labeled compound, forexample a binding partner or antibody having known binding affinity forIL-B60; a source of IL-B60 (naturally occurring or recombinant); and ameans for separating bound from free labeled compound, such as a solidphase for immobilizing the molecule. Once compounds are screened, thosehaving suitable binding affinity to the antigen can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the IL-B60 signalingpathway. The availability of recombinant IL-B60 polypeptides alsoprovide well defined standards for calibrating such assays.

A preferred kit for determining the concentration of, e.g., an IL-B60 ina sample would typically comprise a labeled compound, e.g., bindingpartner or antibody, having known binding affinity for the antigen, asource of cytokine (naturally occurring or recombinant) and a means forseparating the bound from free labeled compound, e.g., a solid phase forimmobilizing the IL-B60. Compartments containing reagents, andinstructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for the IL-B60or fragments are useful in diagnostic applications to detect thepresence of elevated levels of IL-B60 and/or its fragments. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the antigen in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-binding partner complex) or heterogeneous (witha separation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA),enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique(EMIT), substrate-labeled fluorescent immunoassay (SLFIA), and the like.See, e.g., Van Vunakis, et al. (1980) Meth Enzymol. 70:1-525; Harlow andLane (1980) Antibodies: A Laboratory Manual, CSH Press, NY; and Coligan,et al. (eds. 1993) Current Protocols in Immunology, Greene and Wiley,NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against an IL-B60, as such may be diagnostic of variousabnormal states. For example, overproduction of IL-B60 may result inproduction of various immunological reactions which may be diagnostic ofabnormal physiological states, particularly in proliferative cellconditions such as cancer or abnormal activation or differentiation.Moreover, the distribution pattern available provides information thatthe cytokine is expressed in pancreatic islets, suggesting thepossibility that the cytokine may be involved in function of that organ,e.g., in a diabetes relevant medical condition.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or binding partner, or labeledIL-B60 is provided. This is usually in conjunction with other additives,such as buffers, stabilizers, materials necessary for signal productionsuch as substrates for enzymes, and the like. Preferably, the kit willalso contain instructions for proper use and disposal of the contentsafter use. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification or may be modified ina variety of ways. For example, labeling may be achieved by covalentlyor non-covalently joining a moiety which directly or indirectly providesa detectable signal. In any of these assays, the binding partner, testcompound, IL-B60, or antibodies thereto can be labeled either directlyor indirectly. Possibilities for direct labeling include label groups:radiolabels such as ¹²⁵I, enzymes such as peroxidase and alkalinephosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable ofmonitoring the change in fluorescence intensity, wavelength shift, orfluorescence polarization. Possibilities for indirect labeling includebiotinylation of one constituent followed by binding to avidin coupledto one of the above label groups.

There are also numerous methods of separating the bound from the freeIL-B60, or alternatively the bound from the free test compound. TheIL-B60 can be immobilized on various matrixes followed by washing.Suitable matrixes include plastic such as an ELISA plate, filters, andbeads. See, e.g., Coligan, et al. (eds. 1993) Current Protocols inImmunology, Vol. 1, Chapter 2, Greene and Wiley, NY. Other suitableseparation techniques include, without limitation, the fluoresceinantibody magnetizable particle method described in Rattle, et al. (1984)Clin. Chem. 30:1457-1461, and the double antibody magnetic particleseparation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence ofan IL-B60. These sequences can be used as probes for detecting levels ofthe IL-B60 message in samples from patients suspected of having anabnormal condition, e.g., inflammatory or autoimmune. Since the cytokinemay be a marker or mediator for activation, it may be useful todetermine the numbers of activated cells to determine, e.g., whenadditional therapy may be called for, e.g., in a preventative fashionbefore the effects become and progress to significance. The preparationof both RNA and DNA nucleotide sequences, the labeling of the sequences,and the preferred size of the sequences has received ample descriptionand discussion in the literature. See, e.g., Langer-Safer, et al. (1982)Proc. Nat'l. Acad. Sci. 79:4381-4385; Caskey (1987) Science 236:962-967;and Wilchek et al. (1988) Anal. Biochem. 171:1-32.

Diagnostic kits which also test for the qualitative or quantitativeexpression of other molecules are also contemplated. Diagnosis orprognosis may depend on the combination of multiple indications used asmarkers. Thus, kits may test for combinations of markers. See, e.g.,Viallet, et al. (1989) Progress in Growth Factor Res. 1:89-97. Otherkits may be used to evaluate other cell subsets.

X. Isolating a IL-B60 Receptor

Having isolated a ligand of a specific ligand-receptor interaction,methods exist for isolating the receptor. See, Gearing, et al. (1989)EMBO J. 8:3667-3676. For example, means to label the IL-B60 cytokinewithout interfering with the binding to its receptor can be determined.For example, an affinity label can be fused to either the amino- orcarboxyl-terminus of the ligand. Such label may be a FLAG epitope tag,or, e.g., an Ig or Fc domain. An expression library can be screened forspecific binding of the cytokine, e.g., by cell sorting, or otherscreening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271; and Liu, et al. (1994) J. Immunol. 152:1821-29.Alternatively, a panning method may be used. See, e.g., Seed and Aruffo(1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369.

Protein cross-linking techniques with label can be applied to isolatebinding partners of the IL-B60 cytokine. This would allow identificationof proteins which specifically interact with the cytokine, e.g., in aligand-receptor like manner.

Early experiments will be performed to determine whether the known IL-6or G-CSF receptor components are involved in response(s) to IL-B60. Itis also quite possible that these functional receptor complexes mayshare many or all components with an IL-B60 receptor complex, either aspecific receptor subunit or an accessory receptor subunit.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

EXAMPLES

I. General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSHPress, NY; Ausubel, et al. Biology Greene Publishing Associates,Brooklyn, N.Y.; Ausubel, et al. (1987 and Supplements) Current Protocolsin Molecular Biology Wiley/Greene, NY.; Innis, et al. (eds. 1990) PCRProtocols: A Guide to Methods and Applications Academic Press, NY;Bonifacino, et al. Current Protocols in Cell Biology Wiley, NY.; andDoyle, et al. Cell and Tissue Culture: Laboratory Protocols Wiley, NY.Methods for protein purification include such methods as ammoniumsulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; Coligan, et al. (1995 and supplements) Current Protocols inProtein Science John Wiley and Sons, New York, N.Y.; Matsudaira (ed.1993) A Practical Guide to Protein and Peptide Purification forMicrosequencing, Academic Press, San Diego, Calif.; and manufacturer'sliterature on use of protein purification products, e.g., Pharmacia,Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combination withrecombinant techniques allow fusion to appropriate segments (epitopetags), e.g., to a FLAG sequence or an equivalent which can be fused,e.g., via a protease-removable sequence. See, e.g., Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, PlenumPress, NY; and Crowe, et al. (1992) QIAexpress: The High LevelExpression & Protein Purification System QUIAGEN, Inc., Chatsworth,Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the University of Wisconsin GeneticsComputer Group (GCG), Madison, Wis., the NCBI at NIH, and GenBank, NCBI,EMBO, and other sources of public sequence. Other analysis sourcesinclude, e.g., RASMOL program, see Bazan, et al. (1996) Nature 379:591;Lodi, et al. (1994) Science 263:1762-1766; Sayle and Milner-White (1995)TIBS 20:374-376; and Gronenberg, et al. (1991) Protein Engineering4:263-269; and DSC, see King and Sternberg (1996) Protein Sci.5:2298-2310. See, also, Wilkins, et al. (eds. 1997) Proteome Research:New Frontiers in Functional Genomics Springer-Verlag, NY.; Salzberg, etal. (eds. 1998) Computational Methods in Molecular Biology Elsevier,NY.; and Birren, et al. (eds. 1997) Genome Analysis: A Laboratory ManualCold Spring Harbor Press, Cold Spring Harbor, N.Y.

Standard immunological techniques are described, e.g., in Hertzenberg,et al. (eds. 1996) Weir's Handbook of Experimental Immunology vols. 1-4,Blackwell Science; Coligan (1991 and updates) Current Protocols inImmunology Wiley/Greene, NY.; and Methods in Enzymology vols. 70, 73,74, 84, 92, 93, 108, 116, 121, 132, 150, 162, and 163. Cytokine assaysare described, e.g., in Thomson (ed. 1994) The Cytokine Handbook (2ded.) Academic Press, San Diego; Metcalf and Nicola (1995) TheHematopoietic Colony Stimulating Factors Cambridge University Press; andAggarwal and Gutterman (1991) Human Cytokines Blackwell Pub.

Assays for vascular biological activities are well known in the art.They will cover angiogenic and angiostatic activities in tumor, or othertissues, e.g., arterial smooth muscle proliferation (see, e.g., Koyoma,et al. (1996) Cell 87:1069-1078), monocyte adhesion to vascularepithelium (see McEvoy, et al. (1997) J. Exp. Med. 185:2069-2077), etc.See also Ross (1993) Nature 362:801-809; Rekhter and Gordon (1995) Am.J. Pathol. 147:668-677; Thyberg, et al. (1990) Atherosclerosis10:966-990; and Gumbiner (1996) Cell 84:345-357.

Assays for neural cell biological activities are described, e.g., inWouterlood (ed. 1995) Neuroscience Protocols modules 10, Elsevier;Methods in Neurosciences Academic Press; and Neuromethods Humana Press,Totowa, N.J. Methodology of developmental systems is described, e.g., inMeisami (ed.) Handbook of Human Growth and Developmental Biology CRCPress; and Chrispeels (ed.) Molecular Techniques and Approaches inDevelopmental Biology Interscience.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Cloning of Human IL-B60

The sequence of the gene is provided in Table 1. The sequence is derivedfrom a genomic human sequence. These sequences allow preparation of PCRprimers, or probes, to determine cellular distribution of the gene. Thesequences allow isolation of genomic DNA which encode the message.

Using the probe or PCR primers, various tissues or cell types are probedto determine cellular distribution. PCR products are cloned using, e.g.,a TA cloning kit (Invitrogen). The resulting cDNA plasmids are sequencedfrom both termini on an automated sequencer (Applied Biosystems).

III. Cellular Expression of IL-B60

An appropriate probe or primers specific for cDNA encoding primateIL-B60 are prepared. Typically, the probe is labeled, e.g., by randompriming. The expression is probably in the cell types described, andperhaps also in pancreatic islets.

The presence of a leader sequence led to the expectation of findingIL-B60 secreted when expressed in mammalian cells. Transfection of 293Tcells with a tagged form of hIL-B60 (hIL-B60-Etag) did not result inefficient secretion of IL-B60. Instead, IL-B60 could only beimmunoprecipitated from the lysate of the transfected cells. Thepossibility was investigated of IL-B60 being a composite factor likeIL-12 (p35/p40) and thus needing a partner for secretion. Among thenon-signaling receptors of the IL-6 family the recently described and,thus far, orphan, receptor CLF-1 (NR6) also showed high level ofhomology between human and murine forms (>95% amino acid identity).Based on these observations a hypothesis was generated that the IL-B60and CLF-1 are partners.

Southern Analysis: DNA (5 μg) from a primary amplified cDNA library isdigested with appropriate restriction enzymes to release the inserts,run on a 1% agarose gel and transferred to a nylon membrane (Schleicherand Schuell, Keene, N.H.).

Samples for human mRNA isolation can include, e.g., peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T111); Tcell tumor lines Jurkat and Hut78, resting (T117); T cell clones, pooledAD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cellrandom γδ T cell clones, resting (T119); CD28-T cell clone; Splenocytes,resting (B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); Bcell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 hpooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106);hematopoietic precursor line TF1, activated with PMA and ionomycin for1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled(M101); elutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated withLPS, IFNγ, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriatedmonocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 h pooled(M106); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 4, 16h pooled (M107); elutriated monocytes, activated LPS for 1 h (M108);elutriated monocytes, activated LPS for 6 h (M109); DC 70% CD1a+, fromCD34+GM-CSF, TNFα 12 days, resting (D101); DC 70% CD1a+, fromCD34+GM-CSF, TNFα 12 days, activated with PMA and ionomycin for 1 hr(D102); DC 70% CD1a+, from CD34+GM-CSF, TNFα 12 days, activated with PMAand ionomycin for 6 hr (D103); DC 95% CD1a+, from CD34+ GM-CSF, TNFα 12days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled(D104); DC 95% CD14+, ex CD34+ GM-CSF, TNFα 12 days FACS sorted,activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CD1a+ CD86+,from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMA andionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5days, resting (D107); DC from monocytes GM-CSF, IL-4 5 days, resting(D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 hpooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFα,monocyte supe for 4, 16 h pooled (D110); epithelial cells, unstimulated;epithelial cells, IL-1β activated; lung fibroblast sarcoma line MRC5,activated with PMA and ionomycin for 1, 6 h pooled (C101); kidneyepithelial carcinoma cell line CHA, activated with PMA and ionomycin for1, 6 h pooled (C102). Expression of IL-B60 transcript was very high inelutriated monocytes, activated with LPS, IFNγ, anti-IL-10 for 4, 16 hpooled (M106); elutriated monocytes, activated with LPS, IFNγ,anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes,activated LPS for 6 h (M109); and elutriated monocytes, activated LPSfor 1 h (M108).

Samples for mouse mRNA expression can include, e.g., resting mousefibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogenreceptor) transfected cells, control (C201); Mel14+ naive T cells fromspleen, resting (T209); Mel14+ naive T cells from spleen, stimulatedwith IFNγ, IL-12, and anti IL-4 to polarize to TH1 cells, exposed toIFNγ and IL-4 for 6, 12, 24 h, pooled (T210); Mel14+ naive T cells fromspleen, stimulated with IL-4 and anti IFNγ to polarize to Th2 cells,exposed to IL-4 and anti IFNγ for 6, 13, 24 h, pooled (T211); T cells,TH1 polarized (Mel14 bright, CD4+ cells from spleen, polarized for 7days with IFN-γ and anti IL-4; T200); T cells, TH2 polarized (Mel14bright, CD4+ cells from spleen, polarized for 7 days with IL-4 andanti-IFN-γ; T201); T cells, highly TH1 polarized 3× from transgenicBalb/C (see Openshaw; et al. (1995) J. Exp. Med. 182:1357-1367;activated with anti-CD3 for 2, 6, 24 h pooled; T202); T cells, highlyTH2 polarized 3× from transgenic Balb/C (activated with anti-CD3 for 2,6, 24 h pooled (T203); T cells, highly TH1 polarized 3× from transgenicC57 bl/6 (activated with anti-CD3 for 2, 6, 24 h pooled; T212); T cells,highly TH2 polarized 3× from transgenic C57 bl/6 (activated withanti-CD3 for 2, 6, 24 h pooled; T213); T cells, highly TH1 polarized(naive CD4+ T cells from transgenic Balb/C, polarized 3× with IFNγ,IL-12, and anti-IL-4; stimulated with IGIF, IL-12, and anti IL-4 for 6,12, 24 h, pooled); CD44-CD25+ pre T cells, sorted from thymus (T204);TH1 T cell clone D1.1, resting for 3 weeks after last stimulation withantigen (T205); TH1 T cell clone D1.1 , 10 μg/ml ConA stimulated 15 h(T206); TH2 T cell clone CDC35, resting for 3 weeks after laststimulation with antigen (T207); TH2 T cell clone CDC35, 10 μg/ml ConAstimulated 15 h (T208); unstimulated B cell line CH12 (B201);unstimulated mature B cell leukemia cell line A20 (B200); unstimulatedlarge B cells from spleen (B202); B cells from total spleen, LPSactivated (B203); metrizamide enriched dendritic cells from spleen,resting (D200); dendritic cells from bone marrow, resting (D201);unstimulated bone marrow derived dendritic cells depleted with antiB220, anti CD3, and anti Class II, cultured in GM-CSF and IL-4 (D202);bone marrow derived dendritic cells depleted with anti B220, anti CD3,and anti Class II, cultured in GM-CSF and IL-4, stimulated with antiCD40 for 1, 5 d, pooled (D203); monocyte cell line RAW 264.7 activatedwith LPS 4 h (M200); bone-marrow macrophages derived with GM and M-CSF(M201); bone-marrow macrophages derived with GM-CSF, stimulated withLPS, IFNγ, and IL-10 for 24 h (M205); bone-marrow macrophages derivedwith GM-CSF, stimulated with LPS, IFNγ, and anti IL-10 for 24 h (M206);peritoneal macrophages (M207); macrophage cell line J774, resting(M202); macrophage cell line J774+LPS+anti-IL-10 at 0.5, 1, 3, 6, 12 hpooled (M203); macrophage cell line J774+LPS+IL-10 at 0.5, 1, 3, 5, 12 hpooled (M204); unstimulated mast cell lines MC-9 and MCP-12 (M208);immortalized endothelial cell line derived from brain microvascularendothelial cells, unstimulated (E200); immortalized endothelial cellline derived from brain microvascular endothelial cells, stimulatedovernight with TNFα (E201); immortalized endothelial cell line derivedfrom brain microvascular endothelial cells, stimulated overnight withTNFα (E202); immortalized endothelial cell line derived from brainmicrovascular endothelial cells, stimulated overnight with TNFα andIL-10 (E203); total aorta from wt C57 bl/6 mouse; total aorta from 5month ApoE KO mouse (X207); total aorta from 12 month ApoE KO mouse(X207); wt thymus (0214); total thymus, rag-1 (0208); total kidney,rag-1 (0209); total kidney, NZ B/W mouse; and total heart, rag-1 (0202).

The human IL-B60 was found expressed in T cells; the Th0 clone Mot72(activated); activated PBL; monocytes; dendritic cells; fetal lung, andheavy smoker lung samples.

The CLF-1 was found expressed in dendritic cells; splenocytes; Th1cells; fetal lung; and lung samples. This distribution is consistentwith the complex being important in immune function, e.g., dendritic andimmune cells, and in lung physiology.

Since CLF-1 is necessary for IL-B60 secretion in vitro, various humanand mouse cDNA libraries were screened for co-expression of both mRNAs.Highest expression for both was found in adult human splenocytes, Tcells, activated monocytes and dendritic cells and in fetal lung, anduterus. In mouse libraries, co-expression was strongest in adult lung.

IV. Chromosome Mapping of IL-B60

An isolated cDNA encoding the IL-B60 is used. Chromosome mapping is astandard technique. See, e.g., BIOS Laboratories (New Haven, Conn.) andmethods for using a mouse somatic cell hybrid panel with PCR.

The human IL-B60 gene has been localized to human chromosome 11.

V. Purification of IL-B60 Protein or Complexes

Multiple transfected cell lines are screened for one which expresses thecytokine at a high level compared with other cells. Alternatively, arecombinant construct with both subunits can be made. Various cell linesare screened and selected for their favorable properties in handling.Natural IL-B60 can be isolated from natural sources, or by expressionfrom a transformed cell using an appropriate expression vector.Purification of the expressed protein or complex is achieved by standardprocedures, or may be combined with engineered means for effectivepurification at high efficiency from cell lysates or supernatants. FLAGor His6 segments can be used for such purification features.Alternatively, affinity chromatography may be used with specificantibodies, see below.

Protein is produced in coli, insect cell, or mammalian expressionsystems, as desired.

VI. Isolation of Homologous IL-B60 Genes

The IL-B60 cDNA, or other species counterpart sequence, can be used as ahybridization probe to screen a library from a desired source, e.g., aprimate cell cDNA library. Many different species can be screened bothfor stringency necessary for easy hybridization, and for presence usinga probe. Appropriate hybridization conditions will be used to select forclones exhibiting specificity of cross hybridization.

Screening by hybridization using degenerate probes based upon thepeptide sequences will also allow isolation of appropriate clones.Alternatively, use of appropriate primers for PCR screening will yieldenrichment of appropriate nucleic acid clones.

Similar methods are applicable to isolate either species, polymorphic,or allelic variants. Species variants are isolated using cross-specieshybridization techniques based upon isolation of a full length isolateor fragment from one species as a probe.

Alternatively, antibodies raised against human IL-B60 will be used toscreen for cells which express cross-reactive proteins from anappropriate, e.g., cDNA library. The purified protein or definedpeptides are useful for generating antibodies by standard methods, asdescribed above. Synthetic peptides or purified protein are presented toan immune system to generate monoclonal or polyclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; andHarlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. The resulting antibodies are used for screening,purification, or diagnosis, as described.

VII. Antibodies Specific for IL-B60 or Complexes

Synthetic peptides or purified protein are presented to an immune systemto generate monoclonal or polyclonal antibodies. See, e.g., Coligan(1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press.Polyclonal serum, or hybridomas may be prepared. In appropriatesituations, the binding reagent is either labeled as described above,e.g., fluorescence or otherwise, or immobilized to a substrate forpanning methods. Immunoselection, immunodepletion, and relatedtechniques are available to prepare selective reagents, as desired,e.g., for the IL-B60 alone, or the complex between the two subunits.

VIII. IL-B60 and CLF-1 Coprecipitate

A CLF-1-FLAG construct was prepared in an expression vector. AnIL-B60Etag (epitope tagged) construct was also prepared. Transienttransfection into COS cells either with the IL-B60Etag construct alone,the CLF-1-FLAG construct alone, or both together. Cells were labeledwith ³⁵S methionine. The supernatants and cells were collected.

Upon co-transfection of cells with IL-B60-Etag and soluble receptorCLF-1-Flag, secretion of both ligand and soluble receptor was greatlyenhanced. Both could be immunoprecipitated with antibodies againsteither the ligand (anti Etag) or the receptor (anti Flag), indicatingthat IL-B60 and CLF-1 form a soluble cytokine/receptor complex similarto IL-12 (p35/p40). See Gubler, et al. (1991) Proc. Nat'l Acad. Sci. USA88:4143-4147; Wolf, et al. (1991) J. Immunol. 146:3074-3081. Thus,coexpression with a correct partner will result in a dramatic increasein the secretion of the gene products. Coexpression of IL-B60 with othersoluble receptors including Ebi3 (Devergne, et al. (1996) J. Virol.70:1143-1153), IL-12 p40, and sCNTFR (Davis, et al. (1991) Science253:59-63) did not result in efficient secretion of the ligand.

The supernatants were immunoprecipitated with either anti-FLAG M2(precipitates CLF-1) or anti-Etag Ab (precipitates IL-B60). InIL-B60Etag transfectants alone, the level of expression in thesupernatant detected using the antiEtag antibodies was very low. Incontrast, in the double transfectants, the IL-B60Etag and a secondlabeled band were immunoprecipitated. The second band corresponds to theCLF-1. Thus, the Etag antibody immunoprecipitates both proteins, e.g.,they form a complex. In the single transfectant CLF-1FLAG, a little bitof CLF-1FLAG protein is immunoprecipitated with the anti-FLAG M2 Ab.This result is consistent with the other soluble receptors, e.g., forp40 component of IL-12. However, in the double transfectants not only ismore CLF-1 seen, but now also IL-B60. The immunoprecipitation works inboth directions.

IX. IL-B60 Binds to the CNTFR

To identify the signaling receptors for IL-B60/CLF-1 conditioned mediumfrom hIL-B60 and mCLF-1 cotransfected 293T cells was added to BA/F3cells stably transfected with human gp130 alone or hgp130 in combinationwith the hIL-6R, hOSMR, hLIFR, or hLIFR and hCNTFR, respectively. OnlyBA/F3 cells expressing gp130, LIFR, and CNTFR showed a proliferativeresponse upon stimulation with IL-B60/CLF-1. To analyze the possibilityof a signaling complex consisting of CNTFR/gp130 or CNTFR/LIFR only, twosoluble fusion proteins were designed connecting either the CNTFR orCLF-1 to IL-B60 via a flexible linker. Similar so-called hyper-cytokineshave been shown to be 100-1000× more active on cells than cytokine andsoluble receptor added separately. See Fischer, et al. (1997) NatureBiotechnol. 15:142-145. Hyper-CNTFR-IL-B60 was able to induceproliferation of BAF3/gp130/LIFR cells but not of BAF3/gp130 cells,showing that the LIFR is a component of the signaling complex.Stimulation of cells with hyper-CLF-1-IL-B60 did not result inproliferation of any cell line. This indicated that although necessaryfor IL-B60 secretion, CLF-1 is not a subunit of the active signalingreceptor complex.

Involvement of gp130 in the active receptor complex was shown with aneutralizing antibody against gp130 which completely blocked thisresponse. Furthermore, analysis of signal transducers in lysates fromBA/F3 cells expressing gp130, LIFR, and CNTFR showed that STAT3 is onlyphosphorylated after stimulation with either co-expressed IL-B60 andCLF-1 or with the CNTFR-IL-B60 fusion protein but not with theCLF-1-IL-B60 fusion.

X. Evaluation of Breadth of Biological Functions

Biological activities of IL-B60 or complex are tested based, e.g., onthe sequence and structural homology between IL-B60 and IL-6 and G-CSF.Initially, assays that had shown biological activities of IL-6 or G-CSFare examined.

A. Regulation of IL-B60 and CLF-1 After Sciatic Nerve Injury

IL-B60 and CLF-1 expression in the mouse spinal cord was analyzed inunilateral transection of the sciatic nerve followed by separation ofproximal and distal nerve stumps, thus preventing regeneration. Atvarious time points, tissue from the transection area was collected andanalyzed by quantitative PCR for expression of IL-B60 and CLF-1.Transection of the sciatic nerve resulted in fast and long lastingupregulation of ligand and receptor. After 6 hrs IL-B60 and CLF-1 wereupregulated. Expression was still elevated 20 days after transectionwhen compared to non-lesioned or sham-lesioned nerves. In regeneratingaxons (crushed nerves) both IL-B60 and CLF-1 are downregulated after 12h, but whereas IL-B60 expression almost reaches levels of non-lesionednerves after 20 days, CLF-1 levels peak after 20 days. This might pointto an additional function of CLF-1, possibly in remyelination, whichstarts after two weeks. Transection of the sciatic nerve in mice lackingGM-CSF and a macrophage response in nerve shows that IL-B60 expressionafter 4 days is not altered compared to normal mice. However, CLF-1levels in those mice are heterogeneous, with a range from no alterationto an almost 4 fold increase of expression compared to normallittermates.

B. Effects on Proliferation of Cells

The effect on proliferation of various cell types are evaluated withvarious concentrations of cytokine. A dose response analysis isperformed, in combinations with the related cytokines IL-6, G-CSF, etc.A cytosensor machine may be used, which detects cell metabolism andgrowth (Molecular Devices, Sunnyvale, Calif.).

C. Effects on the Expression of Cell Surface Molecules on HumanMonocytes

Monocytes are purified by negative selection from peripheral bloodmononuclear cells of normal healthy donors. Briefly, 3×10⁸ ficoll bandedmononuclear cells are incubated on ice with a cocktail of monoclonalantibodies (Becton-Dickinson; Mountain View, Calif.) consisting, e.g.,of 200 μl of αCD2 (Leu-5A), 200 μl of αCD3 (Leu-4), 100 μl of αCD8 (Leu2a), 100 μl of αCD19 (Leu-12), 100 μl of αCD20 (Leu-16), 100 μl of αCD56(Leu-19), 100 μl of αCD67 (IOM 67; Immunotech, Westbrook, Me.), andanti-glycophorin antibody (10F7MN, ATCC, Rockville, Md.). Antibody boundcells are washed and then incubated with sheep anti-mouse IgG coupledmagnetic beads (Dynal, Oslo, Norway) at a bead to cell ratio of 20:1.Antibody bound cells are separated from monocytes by application of amagnetic field. Subsequently, human monocytes are cultured in Yssel'smedium (Gemini Bioproducts, Calabasas, Calif.) containing 1% human ABserum in the absence or presence of IL-B60, IL-6, G-CSF or combinations.

Analyses of the expression of cell surface molecules can be performed bydirect immunofluorescence. For example, 2×10⁵ purified human monocytesare incubated in phosphate buffered saline (PBS) containing 1% humanserum on ice for 20 minutes. Cells are pelleted at 200×g. Cells areresuspended in 20 ml PE or FITC labeled mAb. Following an additional 20minute incubation on ice, cells are washed in PBS containing 1% humanserum followed by two washes in PBS alone. Cells are fixed in PBScontaining 1% paraformaldehyde and analyzed on FACScan flow cytometer(Becton Dickinson; Mountain View, Calif.). Exemplary mAbs are used,e.g.: CD11b (anti-mac1), CD11c (a gp150/95), CD14 (Leu-M3), CD54 (Leu54), CD80 (anti-BB1/B7), HLA-DR (L243) from Becton-Dickinson and CD86(FUN 1; Pharmingen), CD64 (32.2; Medarex), CD40 (mAb89; Schering-PloughFrance).

D. Effects of IL-B60 or Complex on Cytokine Production by HumanMonocytes

Human monocytes are isolated as described and cultured in Yssel's medium(Gemini Bioproducts, Calabasas, Calif.) containing 1% human AB serum inthe absence or presence of IL-B60 (1/100 dilution baculovirus expressedmaterial). In addition, monocytes are stimulated with LPS (E. coli0127:B8 Difco) in the absence or presence of IL-B60 and theconcentration of cytokines (IL-1β, IL-6, TNFα, GM-CSF, and IL-10) in thecell culture supernatant determined by ELISA.

For intracytoplasmic staining for cytokines, monocytes are cultured (1million/ml) in Yssel's medium in the absence or presence of IL-B60 andLPS (E. coli 0127:B8 Difco) and 10 mg/ml Brefeldin A (Epicentretechnologies Madison Wis.) for 12 hrs. Cells are washed in PBS andincubated in 2% formaldehyde/PBS solution for 20 minutes at RT.Subsequently cells are washed, resuspended in permeabilization buffer(0.5% saponin (Sigma) in PBS/BSA (0.5%)/Azide (1 mM)) and incubated for20 minutes at RT. Cells (2×10⁵) are centrifuged and resuspended in 20 mldirectly conjugated anti-cytokine mAbs diluted 1:10 in permeabilizationbuffer for 20 minutes at RT. The following antibodies can be used:IL-1α-PE (364-3B3-14); IL-6-PE (MQ2-13A5); TNFα-PE (MAb11); GM-CSF-PE(BVD2-21C11); and IL-12-PE (C11.5.14; Pharmingen San Diego, Calif.).Subsequently, cells are washed twice in permeabilization buffer and oncein PBS/BSA/Azide and analyzed on FACScan flow cytometer (BectonDickinson; Mountain View, Calif.).

E. Effects of IL-B60 on Proliferation of Human Peripheral BloodMononuclear Cells (PBMC)

Total PBMC are isolated from buffy coats of normal healthy donors bycentrifugation through ficoll-hypaque as described (Boyum, et al.). PBMCare cultured in 200 μl Yssel's medium (Gemini Bioproducts, Calabasas,Calif.) containing 1% human AB serum in 96 well plates (Falcon,Becton-Dickinson, N.J.) in the absence or presence of IL-B60. Cells arecultured in medium alone or in combination with 100 U/ml IL-2 (R&DSystems) for 120 hours. 3H-Thymidine (0.1 mCi) is added during the lastsix hours of culture and 3H-Thymidine incorporation determined by liquidscintillation counting.

The native, recombinant, and fusion proteins would be tested for agonistand antagonist activity in many other biological assay systems, e.g., onT-cells, B-cells, NK, macrophages, dendritic cells, hematopoieticprogenitors, etc. Because of the IL-6 and G-CSF structural relationship,assays related to those activities should be analyzed

IL-B60 is evaluated for agonist or antagonist activity on transfectedcells expressing IL-6 or G-CSF receptor and controls. See, e.g., Ho, etal. (1993) Proc. Nat'l Acad. Sci. USA 90, 11267-11271; Ho, et al. (1995)Mol. Cell. Biol. 15:5043-5053; and Liu, et al. (1994). J. Immunol.152:1821-1829.

IL-B60 is evaluated for effect in macrophage/dendritic cell activationand antigen presentation assays, T cell cytokine production andproliferation in response to antigen or allogeneic stimulus. See, e.g.,de Waal Malefyt et al. (1991) J. Exp. Med. 174:1209-1220; de WaalMalefyt et al. (199.1) J. Exp. Med. 174:915-924; Fiorentino, et al.(.1991) J. Immunol. 147, 3815-3822; Fiorentino, et al. (1991) J.Immunol. 146:3444-3451; and Groux, et al. (1996) J. Exp. Med. 184:19-29.

IL-B60 will also be evaluated for effects on NK cell stimulation. Assaysmay be based, e.g., on Hsu, et al. (1992) Internat. Immunol. 4:563-569;and Schwarz, et al. (1994) J. Immunother. 16:95-104.

B cell growth and differentiation effects will be analyzed, e.g., by themethodology described, e.g., in Defrance, et al. (1992). J. Exp. Med.175:671-682; Rousset, et al. (1992) Proc. Nat'l Acad. Sci. USA89:1890-1893; including IgG2 and IgA2 switch factor assays. Note that,unlike COS7 supernatants, NIH3T3 and COP supernatants apparently do notinterfere with human B cell assays.

F. IL-B60 and CLF-1 Induce a Switch in Neurotransmitter Properties

Cholineric sympathetic neurons innervate at least three differenttargets: sweat glands, vasculature in skeletal muscle, and periosteum.Mature innervation of sympathetic neurons begins at the end of the firstpostnatal week and is characterized by the appearance of cholinericproperties. Cultures of sympathetic neurons were analyzed for theinduction of different neuromodulators, which specify the cholinergicphenotype. Cholecystokinin (CCK), vasoactive intestinal polypeptide(VIP), substance P (SP) and somatostatin (SOM) are upregulated afterstimulation of neurons with conditioned medium from IL-B60/CLF-1cotransfected cells or the CNTFR-IL-B60 fusion protein. Thus, thecomplex exhibits significant developmental biology function, and may beeffective in inducing certain aspects of neural development.

XI. Generation and Analysis of Genetically Altered Animals

Transgenic mice can be generated by standard methods. Such animals areuseful to determine the effects of overexpression of the gene, inspecific tissues, or completely throughout the organism. Such mayprovide interesting insight into development of the animal or particulartissues in various stages. Moreover, the effect on various responses tobiological stress can be evaluated. See, e.g., Hogan, et al. (1995)Manipulating the Mouse Embryo: A Laboratory Manual (2d ed.) Cold SpringHarbor Laboratory Press.

Adenovirus techniques are available for expression of the gene invarious cells and organs. See, e.g., Hitt, et al. (1997) Adv. Pharmacol.40:137-195; and literature from Quantum Biotechnologies, Montreal,Canada. Animals may be useful to determine the effects of the gene onvarious developmental or physiologically functional animal systems.

The genomic structure for the mouse IL-B60 has been determined. Astrategy for the production of IL-B60 knock-out mice can be developed,and appropriate constructs made.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. An isolated soluble cytokine complex comprising the mature forms ofSEQ ID NO: 2 and SEQ ID NO:
 12. 2. The cytokine complex of claim 1,wherein the cytokine complex binds to a receptor complex comprisinghuman CTNF-R, human gp130, and human LIF-R.
 3. The cytokine complex ofclaim 1, wherein the cytokine complex is attached to at least onedetection tag.
 4. The cytokine complex of claim 3, wherein the detectiontag is selected from the group consisting of FLAG, His6, and Ig domain.5. A kit comprising the cytokine complex of claim 1, wherein the kitcontains: a) a compartment comprising the cytokine complex; and b)instructions for use or disposal of reagents in the kit.
 6. The cytokinecomplex of claim 1, wherein the cytokine complex induces neuromodulatorsselected from the group consisting of cholecystokinin (CCK), vasoactiveintestinal polypeptide (VIP), substance P (SP), and somatostatin (SOM).7. A composition comprising the cytokine complex of claim 1 and anaqueous carrier.
 8. An isolated soluble cytokine fusion proteincomprising a mature form of SEQ ID NO: 2 fused to a mature form of SEQID NO:
 12. 9. The cytokine fusion protein of claim 8, wherein thecytokine complex binds to a receptor complex comprising human CTNF-R,human gp130, and human LIF-R.
 10. The cytokine fusion protein of claim8, wherein the cytokine complex is attached to at least one detectiontag.
 11. The cytokine fusion protein of claim 10, wherein the detectiontag is selected from the group consisting of FLAG, His6, and Ig domain.12. A kit comprising the cytokine fusion protein of claim 8, wherein thekit contains: a) a compartment comprising the cytokine fusion protein;and b) instructions for use or disposal of reagents in the kit.
 13. Acomposition comprising the cytokine fusion protein of claim 8 and anaqueous carrier.
 14. The cytokine complex of claim 8, wherein thecytokine fusion protein induces neuromodulators selected from the groupconsisting of cholecystokinin (CCK), vasoactive intestinal polypeptide(VIP), substance P (SP), and somatostatin (SOM).